G-protein coupled receptor (gpcr)-based biosensors and uses thereof

ABSTRACT

Provided herein are GPCR-based chemical biosensors that can have a sensing unit, a processing unit, and a response unit that can be used to detect a chemical of interest. Also provided herein are methods of making and using the GPCR-based chemical biosensors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/035,734 filed on Aug. 11, 2014, having the title GPCR-BasedBiosensors for Medium-Chain Fatty Acids, the entirety of which isincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberDP14AP00041 awarded by the Defense Advanced Research Projects Agency.The government has certain rights to this invention.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled 02269231.txt, created on Sep. 3, 2015, andhaving a size of 44120 bytes. The content of the sequence listing isincorporated herein in its entirety.

BACKGROUND

Current techniques for identification of microbially produced chemicals,including biofuels, rely on chromatography-based screening assays. Assuch, these current techniques only allow for the processing of about10² samples per day. Therefore, there exists a need for improvedcompositions and techniques that can allow for greater and moreefficient processing of samples.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 shows one embodiment of a GPCR-based chemical biosensor.

FIGS. 2A and 2B show embodiments of a GPCR-based chemical biosensorhaving an optional amplification unit that can directly (FIG. 2A) orindirectly (FIG. 2B) amplify a signal generated by a sensing unit of theGPCR-based chemical sensor.

FIG. 3 shows one embodiment of a medium throughput system for screeningchemical producing cells using a GPCR-based chemical sensor as describedherein.

FIG. 4 shows one embodiment of an engineered yeast strain containingdeletions of Sst2 and Far1 and one embodiment of a GPCR-based chemicalbiosensor containing a Ste2/α-factor sensing unit schematic. The Ste2GPCR can detect α-factor in the culture medium, the chemical signal canbe transmitted via the yeast mating pathway to the mating pathwaytranscription factor Ste12. Ste12 can activate transcription of greenfluorescent protein (GFP) under control of a mating pathway promoter(Pmating pathway). The GPCR-based chemical sensor strain has the far1and sst2 genes deleted to avoid cell cycle arrest and to reduce thespontaneous rate of GPCR inactivation upon chemical sensing,respectively.

FIG. 5 shows a graph demonstrating Ste2/α-factor sensor dose responsecurves carrying GFP under control of two mating pathway promoters (PFus1and PFig1) from either a single-copy (s) or a multicopy (m) reporterplasmids. All experiments were done in triplicate and the error barsrepresent the standard deviation from the mean.

FIG. 6 shows a graph demonstrating maximum x-fold increase in signalafter activation: PFus1-GFP(m): 50 nM α-factor, PFig1-GFP(m): 75 nMα-factor, PFus1-GFP(s) and PFig1-GFP(s): 100 nM α-factor. P-values,obtained from a two-tailed t test, shown for statistically differentsamples. All experiments were done in triplicate and the error barsrepresent the standard deviation from the mean. All experiments weredone in triplicate and the error bars represent the standard deviationfrom the mean.

FIG. 7 is a cartoon depicting one embodiment of a GPCR-based chemicalbiosensor. Either the OR1 G1 or GPR40 GPCR can detect medium-chain fattyacids in the culture medium, the chemical signal is transmitted via theyeast mating pathway to the mating pathway transcription factor Ste12.Ste12 activates transcription of GFP under control of the PFig1promoter. In addition to deletion of the far1 and sst2 genes, themedium-chain fatty acid sensor strain has the endogenous GPCR Ste2deleted (W303 Δfar1, Δsst2, Δste2).

FIG. 8 shows a graph demonstrating dose response curves for the OR1G1-based sensor (PPY643) with C8, C10, C12, C14 and C16 acids. Allexperiments were done in triplicate and the error bars represent thestandard deviation from the mean.

FIG. 9 shows a graph demonstrating OR1G1-based sensor maximum x-foldincrease in signal after activation with C8 (800 μM), C10 (250 μM) andC12 (500 μM) acids. All experiments were done in triplicate and theerror bars represent the standard deviation from the mean. None of thesamples shows a statistical difference using a two-tailed t test.

FIG. 10 shows a graph demonstrating dose response curves for theGPR40-based sensor (PPY644) with C8, C10, C12, C14 and C16 acids. Allexperiments were done in triplicate and the error bars represent thestandard deviation from the mean.

FIG. 11 shows a graph demonstrating GPR40-based sensor maximum x-foldincrease in signal after activation with C8 (800 μM), C10 (800 μM) andC12 (250 μM) acids. All experiments were done in triplicate and theerror bars represent the standard deviation from the mean. None of thesamples shows a statistical difference using a two-tailed t test.

FIG. 12 shows a cartoon of one embodiment of a GPCR-based chemicalbiosensor having a sensor unit schematic of: chemical sensor strain(W303 Δfar1, Δsst2, Δste2) expressing either OR1G1 or GPR40 carryingPFig1-GFP(s) as the reporter plasmid.

FIG. 13 shows a graph demonstrating GPCR-based sensor signal requiressensing unit (GPCR) for chemical sensing. Chemical sensor strainexpressing no GPCR, OR1G1 or GPR40 in the presence of 0 or 500 μMdecanoic (C10) acid using PFig1-GFP(s) as the reporter plasmid. Allexperiments were done in triplicate and the error bars represent thestandard deviation from the mean.

FIG. 14 shows a graph demonstrating GPCR-based sensor signal requiresresponse unit (Ste12) for chemical sensing. Chemical sensor strain withendogenous yeast mating pathway transcription factor Ste12 deleted (W303Δfar1, Δsst2, Δste2, Δste12) expressing no GPCR, OR1G1 or GPR40 in thepresence of 0 or 500 μM decanoic (C10) acid using PFig1-GFP(s) as thereporter plasmid. P-values, obtained from a two-tailed t test, shown forstatistically different samples. All experiments were done in triplicateand the error bars represent the standard deviation from the mean.

FIGS. 15A-15C show graphs demonstrating dose response curves for theOR1G1-based sensor with C8, C10 and C12 fatty aldehydes (FIG. 15A),alcohols (FIG. 15B) and C10 methyl- and ethyl-esters (FIG. 15C). Allexperiments were done in triplicate and the error bars represent thestandard deviation from the mean.

FIGS. 16A-16C show graphs demonstrating dose response curves for theGPR40-based sensor with C8, C10 and C12 fatty aldehydes (FIG. 16A),alcohols (FIG. 16B) and C10 methyl- and ethyl-esters (FIG. 16C). Allexperiments were done in triplicate and the error bars represent thestandard deviation from the mean.

FIG. 17 shows a cartoon depicting embodiments of GPCR-based chemicalbiosensors having synthetic response units. Synthetic transcriptionfactor (STF)/synthetic promoter composition. AD=activation domain,P=phosphorylation domain, DBD=DNA binding domain. STF1 is composed of aGal4AD, Ste12p and a Gal4DBD. STF1 binds to PGal4(5×), a syntheticpromoter carrying five Gal4 DNA binding sites. STF2 can be composed of aB42AD, Ste12p and a LexADBD. STF2 binds to PLexA(4×), a syntheticpromoter carrying four lexA DNA binding sites.

FIG. 18 shows a cartoon depicting embodiments of a GPCR-based chemicalbiosensor. Schematic of the OR1G1- and GPR40-based sensors using asynthetic response unit (STF/Psynthetic-GFP). In addition to deletion ofthe far1, sst2, and ste2 genes, the chemical sensor strain using asynthetic response unit can also have the endogenous transcriptionfactor Ste12 deleted (W303 Δfar1, Δsst2, Δste2, Δste12).

FIG. 19 shows a graph demonstrating dose response curves for decanoicacid using the OR1G1-based sensor coupled to Ste12/PFig1-GFP(s) (blue),STF1/PGal4(5×)-GFP (black), or STF2/PLexA(4×)-GFP (red) response units.All experiments were done in triplicate and the error bars represent thestandard deviation from the mean. P-values, obtained from a two-tailed ttest, shown for statistically different samples.

FIG. 20 shows a graph demonstrating OR1G1-based sensor maximum x-foldincrease in signal after activation upon addition of decanoic acid whencoupled to Ste12/PFig1-GFP(s): 250 μM C10 acid, STF1/PGal4(5×)-GFP: 800μM C10 acid, and STF2/PLexA(4×)-GFP: 800 μM C10 acid. All experimentswere done in triplicate and the error bars represent the standarddeviation from the mean. P-values, obtained from a two-tailed t test,shown for statistically different samples.

FIG. 21 shows a graph demonstrating dose response curves for decanoicacid using the GPR40-based sensor coupled to Ste12/PFig1-GFP(s) (blue),STF1/PGal4(5×)-GFP (black) or STF2/PLexA(4×)-GFP (red) response units.All experiments were done in triplicate and the error bars represent thestandard deviation from the mean.

FIG. 22 shows a graph demonstrating GPR40-based sensor maximum x-foldincrease in signal after activation upon addition of decanoic acid whencoupled to Ste12/PFig1-GFP(s): 800 μM C10 acid, STF1/PGal4(5×)-GFP: 500μM C10 acid, and STF2/PLexA(4×)-GFP: 500 μM C10 acid. P-values, obtainedfrom a two-tailed t test, shown for statistically different samples. Allexperiments were done in triplicate and the error bars represent thestandard deviation from the mean.

FIG. 23 is a graph demonstrating GFP fluorescence from OR1G1-basedsensors with P_(FIG1)-GFP multi-copy reporter plasmid. The OR1G1 sensorhad the following characteristics: (W303 Δfar1, Δsst2, Δste2,pESC-His3-P_(TEF1)-OR1G1, pESC-Leu2-P_(FIG1)-GFP).

FIG. 24 is a graph demonstrating GFP fluorescence from GPR40-basedsensors with P_(FIG1)-GFP multi-copy reporter plasmid. The GPR40 sensorhad the following characteristics: (W303 Δfar1, Δsst2, Δste2,pESC-His3-P_(TEF1)-GPR40, pESC-Leu2-P_(FIG1)-GFP).

FIG. 25 is a graph demonstrating OR1G1-based sensor decanoic acidresponse curves when the OR1 G1 GPCR is expressed from a multi- or asingle-copy plasmid.

FIG. 26 is a graph demonstrating OR1G1-based sensor decanoic acidresponse curves when signaling through the endogenous yeast Ga subunitexpressed from the chromosome (GPA1), the mammalian olfactory Ga subunitfrom a multi-copy plasmid (G_(olf) (m)), a hybrid yeast/mammalian Gαsubunit composed of GPA1 carrying the five C-terminal amino acids fromG_(olf) from a multi-(GPA1-G_(olf) (m)) or a single-copy plasmid(GPA1-G_(olf) (m)).

FIG. 27 shows a graph demonstrating OR1G1-sensor performance in amixture of fatty acids. Although the OR1G1-sensor is able to detect C10,C14 and C16 acids at 250 μM independently, it is not able to detect themin a mixture of acids.

FIGS. 28A-28C show cell growth of the sensor strain (28A). Fatty acidtoxicity to the sensor strains (28B,C). Importantly, the growth rate ofthe sensor is similar to the wild type strain.

FIGS. 29A-29E show sample flow cytometry histograms of the biosensorstrain when incubated with different compounds when using GPCR-basedsensor OR1G1. A shift in the population when incubating with C8-C12(orange line) was observed. Blue lines indicate the sensor when nocompound is added. Red line indicates cell autofluorescense.

FIGS. 30A-30E Sample flow cytometry histograms of the biosensor strainwhen incubated with different compounds when using GPCR-based sensorGPR40. You can see a shift of the population when incubating withdecanoic acid (orange line). Blue lines are sensor when no compound isadded. Red line is cell autofluorescense.

FIG. 31 shows a graph demonstrating endogenous GPCR (Ste2/alpha-factor)x-fold increase in signal after activation 4 hours after addition of 100nM of α-factor.

FIG. 32 shows a graph demonstrating endogenous GPCR (Ste2/alpha-factor)x-fold increase in signal with 100 nM of α-factor at different timepoints. The fluorescence was measured after 1, 2, 3, and 4 hours.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of molecular biology, microbiology,nanotechnology, organic chemistry, synthetic biology, chemistry,biochemistry, botany and the like, which are within the skill of theart. Such techniques are explained fully in the literature.

DEFINITIONS

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within ·+−·10% of the indicated value, whichever is greater.

As used herein, “control” is an alternative subject or sample used in anexperiment for comparison purposes and included to minimize ordistinguish the effect of variables other than an independent variable.

As used herein, “specifically binds” or “specific binding” refers tobinding that occurs between such paired species such asenzyme/substrate, receptor/agonist or antagonist, antibody/antigen,lectin/carbohydrate, oligo DNA primers/DNA, enzyme or protein/DNA,and/or RNA molecule to other nucleic acid (DNA or RNA) or amino acid,which may be mediated by covalent or non-covalent interactions or acombination of covalent and non-covalent interactions. When theinteraction of the two species produces a non-covalently bound complex,the binding that occurs is typically electrostatic, hydrogen-bonding, orthe result of lipophilic interactions. Accordingly, “specific binding”occurs between a paired species where there is interaction between thetwo which produces a bound complex having the characteristics of anantibody/antigen, enzyme/substrate, DNA/DNA, DNA/RNA, DNA/protein,RNA/protein, RNA/amino acid, receptor/substrate interaction. Inparticular, the specific binding is characterized by the binding of onemember of a pair to a particular species and to no other species withinthe family of compounds to which the corresponding member of the bindingmember belongs. Thus, for example, an antibody preferably binds to asingle epitope and to no other epitope within the family of proteins.

As used herein, “overexpressed” or “overexpression” refers to anincreased expression level of an RNA or protein product encoded by agene as compared to the level of expression of the RNA or proteinproduct in a normal or control cell.

As used herein, “underexpressed” or “underexpression” refers todecreased expression level of an RNA or protein product encoded by agene as compared to the level of expression of the RNA or proteinproduct in a normal or control cell.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into RNA transcripts. In the context ofmRNA and other translated RNA species, “expression” also refers to theprocess or processes by which the transcribed RNA is subsequentlytranslated into peptides, polypeptides, or proteins.

As used herein, gene deletion refers to a mutation introduced into thegenome of an organism that completely or partially removes a physicalportion of the nucleotide sequence for the gene to disrupt theproduction of a gene product generated from that gene or otherwisedisrupts and/or ablates the production of the product of that gene.Deletions can be said to result in gene knockout or knockdown. Deletionscan be homozygous (both or all copies deleted), heterozygous (only oneor less than all copies deleted), or hemizygous.

As used herein, “nucleic acid” and “polynucleotide” generally refer to astring of at least two base-sugar-phosphate combinations and refers to,among others, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.“Polynucleotide” and “nucleic acids” also encompasses such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. For instance, theterm polynucleotide includes DNAs or RNAs as described above thatcontain one or more modified bases. Thus, DNAs or RNAs comprisingunusual bases, such as inosine, or modified bases, such as tritylatedbases, to name just two examples, are polynucleotides as the term isused herein. “Polynucleotide” and “nucleic acids” also includes PNAs(peptide nucleic acids), phosphorothioates, and other variants of thephosphate backbone of native nucleic acids. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, but contain the same bases. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “nucleic acids” or“polynucleotide” as that term is intended herein.

As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid(RNA)” generally refer to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA(small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA),anti-sense RNA, RNAi (RNA interference construct), siRNA (shortinterfering RNA), or ribozymes.

As used herein, “nucleic acid sequence” and “oligonucleotide” alsoencompasses a nucleic acid and polynucleotide as defined above.

As used herein, “DNA molecule” includes nucleic acids/polynucleotidesthat are made of DNA.

As used herein, “wild-type” is the typical form of an organism, variety,strain, gene, protein, or characteristic as it occurs in nature, asdistinguished from mutant forms that may result from selective breedingor transformation with a transgene.

As used herein, “identity,” is a relationship between two or morepolypeptide or polynucleotide sequences, as determined by comparing thesequences. In the art, “identity” also refers to the degree of sequencerelatedness between polypeptide as determined by the match betweenstrings of such sequences. “Identity” can be readily calculated by knownmethods, including, but not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., Ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determineidentity are designed to give the largest match between the sequencestested. Methods to determine identity are codified in publicly availablecomputer programs. The percent identity between two sequences can bedetermined by using analysis software (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, Madison Wis.) that incorporatesthe Needelman and Wunsch (J. Mol. Biol., 1970, 48: 443-453) algorithm(e.g., NBLAST, and XBLAST). The default parameters are used to determinethe identity for the polypeptides or polynucleotides of the presentdisclosure.

As used herein, “heterologous” refers to compounds, molecules,nucleotide sequences (including genes), and polypeptide sequences(including peptides and proteins) that are different in both activity(function) and sequence or chemical structure. As used herein,“heterologous” can also refer to a gene or gene product that is from adifferent organism. for example, a human GPCR can be said to beheterologous when expressed in yeast.

As used herein, “homologue” refers to a polypeptide sequence that sharesa threshold level of similarity and/or identity as determined byalignment of matching amino acids. Two or more polypeptides determinedto be homologues are said to be homologues. Homology is a qualitativeterm that describes the relationship between polypeptide sequences thatis based upon the quantitative similarity.

As used herein, “paralog” refers to a homologue produced via geneduplication of a gene. In other words, paralogs are homologues thatresult from divergent evolution from a common ancestral gene.

As used herein, “orthologues” refers to homologues produced byspeciation followed by divergence of sequence but not activity inseparate species. When speciation follows duplication and one homologuesorts with one species and the other copy sorts with the other species,subsequent divergence of the duplicated sequence is associated with oneor the other species. Such species specific homologues are referred toherein as orthologues.

As used herein, “xenologs” are homologues resulting from horizontal genetransfer.

As used herein, “similarity” is a quantitative term that defines thedegree of sequence match between two compared polypeptide sequences.

As used herein, “cell,” “cell line,” and “cell culture” include progeny.It is also understood that all progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological property, as screened for inthe originally transformed cell, are included.

As used herein, “culturing” refers to maintaining cells under conditionsin which they can proliferate and avoid senescence as a group of cells.“Culturing” can also include conditions in which the cells also oralternatively differentiate.

As used herein, “organism”, “host”, and “subject” refers to any livingentity comprised of at least one cell. A living organism can be assimple as, for example, a single isolated eukaryotic cell or culturedcell or cell line, or as complex as a mammal, including a human being,and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats,dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears,primates (e.g., chimpanzees, gorillas, and humans). “Subject” may alsobe a cell, a population of cells, a tissue, an organ, or an organism,preferably to human and constituents thereof.

As used herein, “gene” refers to a hereditary unit corresponding to asequence of DNA that occupies a specific location on a chromosome andthat contains the genetic instruction for a characteristic(s) ortrait(s) in an organism.

As used herein, the term “recombinant” generally refers to anon-naturally occurring nucleic acid, nucleic acid construct, orpolypeptide. Such non-naturally occurring nucleic acids may includenatural nucleic acids that have been modified, for example that havedeletions, substitutions, inversions, insertions, etc., and/orcombinations of nucleic acid sequences of different origin that arejoined using molecular biology technologies (e.g., a nucleic acidsequences encoding a fusion protein (e.g., a protein or polypeptideformed from the combination of two different proteins or proteinfragments), the combination of a nucleic acid encoding a polypeptide toa promoter sequence, where the coding sequence and promoter sequence arefrom different sources or otherwise do not typically occur togethernaturally (e.g., a nucleic acid and a constitutive promoter), etc.).Recombinant also refers to the polypeptide encoded by the recombinantnucleic acid. Non-naturally occurring nucleic acids or polypeptidesinclude nucleic acids and polypeptides modified by man.

As used herein, “plasmid” as used herein refers to a non-chromosomaldouble-stranded DNA sequence including an intact “replicon” such thatthe plasmid is replicated in a host cell.

As used herein, the term “vector” or is used in reference to a vehicleused to introduce an exogenous nucleic acid sequence into a cell. Avector may include a DNA molecule, linear or circular (e.g. plasmids),which includes a segment encoding a polypeptide of interest operativelylinked to additional segments that provide for its transcription andtranslation upon introduction into a host cell or host cell organelles.Such additional segments may include promoter and terminator sequences,and may also include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from yeast or bacterial genomicor plasmid DNA, or viral DNA, or may contain elements of both.

As used herein, “operatively linked” indicates that the regulatorysequences useful for expression of the coding sequences of a nucleicacid are placed in the nucleic acid molecule in the appropriatepositions relative to the coding sequence so as to effect expression ofthe coding sequence. This same definition is sometimes applied to thearrangement of coding sequences and transcription control elements (e.g.promoters, enhancers, and termination elements), and/or selectablemarkers in an expression vector.

As used herein, “cDNA” refers to a DNA sequence that is complementary toa RNA transcript in a cell. It is a man-made molecule. Typically, cDNAis made in vitro by an enzyme called reverse-transcriptase using RNAtranscripts as templates.

As used herein, the term “transfection” refers to the introduction of anexogenous and/or recombinant nucleic acid sequence into the interior ofa membrane enclosed space of a living cell, including introduction ofthe nucleic acid sequence into the cytosol of a cell as well as theinterior space of a mitochondria, nucleus, or chloroplast. The nucleicacid may be in the form of naked DNA or RNA, it may be associated withvarious proteins or regulatory elements (e.g., a promoter and/or signalelement), or the nucleic acid may be incorporated into a vector or achromosome.

As used herein, “transformation” or “transformed” refers to theintroduction of a nucleic acid (e.g., DNA or RNA) into cells in such away as to allow expression of the coding portions of the introducednucleic acid.

As used herein, “stable expression,” “stable incorporation,” “stabletransfection” and the like refer to the integration of an exogenous geneinto the genome of a host cell, which can allow for long term expressionof the exogenous gene.

As used herein, “transient expression,” “transient transfection,” andthe like refer to the introduction of an exogenous gene into a host cellthat does not result in stable incorporation of the gene into the hostcell.

As used herein “chemical” refers to any molecule, compound, particle, orother substance that can be a substrate for a G-protein coupledreceptor. As such, “chemical” can refer to nucleic acids, proteins,organic compounds, inorganic compounds, etc.

As used herein “biologically coupled” refers to the association of orinteraction between two or more physically distinct molecules, groups ofmolecules compounds, organisms, or particles where the association isdirectly or indirectly mediated between the two or more physicallydistinct molecules, groups of molecules compounds, organisms orparticles via a biologic molecule or compound. This can include directbinding between two biologic molecules and signal transduction pathways.

As used herein, “biological communication” refers to the communicationbetween two or more molecules, compounds, or objects that is mediated bya biologic molecule or biologic interaction.

As used herein, “biologic molecule,” “biomolecule,” and the like referto any molecule that is present in a living organism and includeswithout limitation, macromolecules (e.g. proteins, polysaccharides,lipids, and nucleic acids) as well as small molecules (e.g. metabolitesand other products produced by a living organism).

As used herein, a “biologic interaction” refers to the interactionbetween two biomolecules.

As used herein, “regulation” refers to the control of gene or proteinexpression or function.

As used herein, “signaling molecule” refers to a molecule, such as abiomolecule, capable of producing a measurable signal when expressed.The signal can be qualitative or quantitative. The signal can bemeasured by any suitable techniques, which will be instantly appreciatedby those of skill in the art.

As used herein, “promoter” refers to the DNA sequence(s) that control orotherwise modify transcription of a gene and can include binding sitesfor transcription factors, RNA polymerases, and other biomolecules andsubstances (e.g. inorganic compounds) that can influence transcriptionof a gene by interaction with the promoter. Typically these sequencesare located at the 5′ end of the sense strand of the gene, but can belocated anywhere in the genome.

As used herein, “native” refers to the endogenous version of a moleculeor compound relative to the host cell or population being described.

As used herein, “non-naturally occurring” refers to a non-native versionof a molecule or compound or non-native expression or presence of amolecule or compound within a host cell or other composition. This caninclude where a native molecule or compound is influenced to beexpressed or present at a different location within a host, at anon-native period of time within a host, or is otherwise in an alteredenvironment, even when considered within the host. Non-limiting examplesinclude where a protein that is expressed only in the nucleus of a cellis expressed in the cytoplasm of the cell or when a protein that is onlynormally expressed during the embryonic stage of development isexpressed during the adult stage.

As used herein, “encode” refers to the biologic phenomena oftranscribing DNA into an RNA that, in some cases, can be translated intoa protein product. As such, when a protein is said herein to be encodedby a particular nucleotide sequence, it is to be understood that thisrefers to this biologic relationship between DNA and protein. It is wellestablished that RNA can be translated into protein based on the tripletcode where 3 nucleotides represent an amino acid. This term alsoincludes the idea that DNA can be transcribed into RNA molecules withbiologic functions, such as ribozymes and interfering RNA species. Assuch, when a RNA molecule is said to be encoded by a particularnucleotide sequence it is to be understood that this is referring to thetranscriptional relationship between the DNA and RNA species inquestion. As such “encoding nucleotide” refers to herein as thenucleotide which can give rise through transcription, and in the case ofproteins, translation a functional RNA or protein.

As used herein, “fast maturing” refers to a signal molecule (e.g. afluorescent protein) that can be measured, preferably within the linearrange of the signal molecule, within about 0 to about 4 hours of initialcontact of the signal molecule, a sensor system containing the signalmolecule (such as those described herein), or sensor organism containingthe signal molecule (such as those described herein), with a sample,substrate of the signaling molecule, aforementioned system or organism.

DISCUSSION

Production of chemicals by microbial bioreactors can provide asustainable, cost-effective, and environmentally friendly alternative tothe synthesis of fuels and other chemicals from petroleum and othernatural products. Identification of most microbially-produced chemicals,including biofuels, currently relies on low-throughput (10² samples perday) chromatography-based screens due to the lack of a chemical handlethat can be exploited for rapid colorimetric detection. Colorimetricmicrobially-produced chemicals, such as lycopene and indigo, areamenable to high-throughput (10⁷ samples per day) screening and havebeen successfully linked to genome engineering strategies for improvedmicrobial production. However, the number chemicals for which acolorimetric assay exists are extremely limited and far eclipsed by thenumber of chemicals for which no such assay exists. Further, many of thecolorimetric assays have limited sensitivity and can only detect a typeof chemical (e.g. fatty acid) as opposed to a specific chemical (e.g.decanoic acid).

To make non-colorimetric chemicals similarly amenable to high-throughputscreening and specific for a particular chemical, improved chemicalsensors are needed. Given the number of microbially-produced chemicalsof interest, these sensors should be capable of rapid assembly fromexisting biological parts. Most chemical biosensors, often encoded byRNA or protein, are composed of a single biological part with twodistinct functional units physically and directly linked to one another.These biosensors typically have a sensing unit to detect the chemicaland an actuator unit to trigger a cellular process, such as proteinfluorescence. An example of this type of biosensor is a fluorescentprotein engineered to bind a chemical and binding of the chemical causesa conformational change in the fluorescent protein, which results in achange in the fluorescence produced by the fluorescent protein.

While single-part RNA- and transcription factor-based sensors have beenapplied to improve the microbial production of chemicals, none rely onallosteric regulation to generate a response to a chemical or theabsence thereof. Efficiently transmitting chemical sensing informationfrom the sensing unit to the actuator unit in single-part sensors ischallenging, as the conformational change between these units must beextensively fine-tuned to effectively and efficiently transition betweenthe on- and off states. This fine-tuning often requires a combination ofin vitro and in vivo screening to engineer a single-part sensor for eachchemical of interest.

With that said, described herein are chemical biosensors that can usetwo different biological parts: one part that is specialized in chemicalsensing and another part this is specialized in actuating the signal,where information is transmitted from the sensing unit to the actuatingunit not via a physical linkage, but via a physically independentprocessing unit. In short, the chemical biosensors described herein canbe rapidly changed in a modular fashion to quickly generate a desiredchemical biosensor. The multi-part chemical biosensors described hereincan overcome the need for extensive fine-tuning of each chemical sensorand allow for the rapid development of chemical biosensors that can beused to screen for chemicals in high-throughput systems.

Other compositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

GPCR-Based Chemical Biosensors

Described herein are GPCR-based chemical biosensors that can containphysically distinct components that are operatively coupled to eachother and can detect a chemical and generate a signal indicating thepresence or absence of a chemical. With the general description in mind,attention is direct to FIG. 1 which shows one embodiment of a GPCR-basedchemical sensor described herein. The GPCR-based chemical sensors 1000described herein can contain a sensing unit 1100, a processing unit1200, and a response unit 1300, where the sensing unit 1100 can bebiologically coupled to and/or in biologic communication with theprocessing unit 1200 and the processing unit can be biologically coupledto and/or in biologic communication with the response unit 1300.

The sensing unit 1100 can contain a GPCR that can bind to or otherwiseinteract with a chemical 1400. The sensing unit 1100 can biologicallyinteract with processing unit 1200, which in turn can biologicallyinteract with the response unit 1300. The biologic interaction betweenthe different units of the GPCR-based chemical biosensors 1000 can bedirect (i.e. no intermediate molecules, processes, and/or pathwaysinvolved in the biological interaction between one or more components ofthe interacting units) or indirect (i.e. involve one or moreintermediate molecules, processes, and/or pathways in the biologicalinteraction between one or more components of the interacting units,where the additional molecules, process and/or pathways are not part ofthe sensing unit, processing unit, or response unit).

In operation, a chemical 1400 can bind, unbind, or otherwise interactwith the GPCR of the sensing unit 1100. Upon chemical interaction withthe GPCR of the sensing unit 1100 the GPCR can biologically interactwith the processing unit 1200. In some embodiments, chemical binding (orother interaction) with the GPCR of the sensing unit 1100 can stimulate,either directly or indirectly, a signal transduction pathway that ispart of the processing unit 1200. The signal transduction pathway of theprocessing unit 1200 can then biologically interact with the responseunit 1300, which can then generate or extinguish a signal. In someembodiments, the biological interaction between the processing unit 1200and the response unit 1300 can be direct or indirect regulation of asignaling molecule gene promoter. In this way the processing unit 1200can transmit a biological signal indicating the interaction of achemical with the sensing unit 1100 to the response unit 1300, which cansignal the presence (or absence) of a chemical 1400.

As shown in FIGS. 2A and 2B, the GPCR-based chemical biosensor 1000 canoptionally contain an amplification unit 2000. The amplification unit2000 can be configured to directly (FIG. 2A) or indirectly (FIG. 2B)amplify the signal generated by the response unit 1300. Generally, theamplification unit 2000 can act as a feed forward loop that stimulatesincreased signal production from the response unit 1300 when theresponse unit is biologically acted upon, either directly or indirectly,by the processing unit 1200. In short, the amplification unit canautoamplify the signal from the response unit 1300 in response to achemical 1400 binding, unbinding, or otherwise interacting with the GPCRof the sensing unit 1100. The mechanism by which amplification can occuris described in greater detail elsewhere herein.

The physically distinct components can be expressed within a whole cell,such as a yeast cell. In other embodiments, the physically distinctcomponents can be expressed in a synthetic in vitro system. Thephysically distinct components can be considered modular componentswhere each one can be independently manipulated and changed withoutalteration of the other components. This modular configuration can allowfor efficient and rapid tuning and customization of system based on thedesired sensing and signaling capabilities of the GPCR-based chemicalbiosensor. The individual modular components are discussed in furtherdetail below.

Sensing Unit

As shown in FIG. 1, the sensing unit 1100 can contain a GPCR. The GPCRcan be a native GPCR. In some embodiments, the GPCR can be anon-naturally occurring GPCR, which includes but is not limited to,recombinant and other engineered GPCRs. In some embodiments, the GPCRcan be a heterologous GPCR, a homologous GPCR, an orthologous GPCR, or aparalogous GPCR. The GPCR can be a GPCR in the family of Rhodopsin-likeGPCRs, Secretin receptor GPCRs, metabotropic glutamate/pheromone GPCRs,fungal mating pheromone GPCRs, Cyclic AMP GPRCs, or Frizzled, SmoothenedGPCRs. In some embodiments, the GPCR is codon optimized for the organismin which the GPCR-based biosensor is to be expressed in. In someembodiments, the GPCR can be GPCR40 (also referred to herein as GPR40).In other embodiments, the GPCR can be OR1G1 or any other olfactoryreceptor GPCR. In other embodiments, the GPCR can be M3 muscarinicreceptor, D2S Dopamine receptor, Beta2 Adrenergic receptor, Beta Alaninereceptor, Nicotinamide receptor, OR56, Geosmin GPCR, melatonin receptor(mella), or AT1R. In further embodiments, the GPCR can be STE2 GPCR orSTE3 GPCR. In some embodiments, the GPCR can have an encoding nucleotidesequence according to any of SEQ ID NOs: 59-75.

The sensing unit 1100 and/or the GPCR of the sensing unit 1100 can beconfigured to biologically interact with the processing unit 1200. TheGPCR can be configured to interact with one or more signal transductionpathways within the host cell (i.e. the cell in which the GPCR isexpressed in). GPCRs contain three subunits (typically denoted Gα,Gβ/Gλ) that interact with each other either by the subunits associatingwith one another upon binding/unbinding a substrate (e.g. chemical) orone or more subunits disassociating from the other subunit(s) uponbinding/unbinding a substrate. The disassociation or association of oneor more subunits of the GPCR can stimulate or inactivate a downstreamsignal transduction pathway present in a host cell or in vitroenvironment. In some embodiments, the signal transduction pathway can bepart of the processing unit of the GPCR-based chemical biosensor. Inother embodiments, this signal transduction pathway is an intermediatebetween the sensing unit 1100 and the processing unit 1200. It will beappreciated by those of ordinary skill in the art that the signaltransduction pathway will vary based on the GPCR employed in the sensingunit 1100 and the host cell. The signal transduction pathway can be aMAPK pathway, adenylyl cyclase pathway, phospholipase C pathway,arachidonic acid pathway, cyclic AMP (cAMP) pathway, RhoGEF signalingpathways, ion channels (e.g. G-protein-regulated rectifying K+ channels,P/Q- and N-type voltage gated channels, and posphoinositide-3-kinasepathways. In some embodiments, the GPCR can directly signal theprocessing unit through signaling β-arrestin, G protein-coupled receptorkinases, and tyrosine kinases (e.g. proto-oncogene tyrosine-proteinkinase Src).

The GPRC of the sensing unit 1100 can be configured to bind any desiredchemical. The GPCR can naturally bind a chemical of interest or can bemodified to have improved or otherwise altered binding characteristics(e.g. bind a substrate that would not naturally bind to the GPCR). Insome embodiments, the GPCR can bind a medium chain (i.e. a C8-C14) fattyacid. In some embodiments, the chemical can be C10 fatty acid.

Processing Unit

The GPCR-based chemical biosensor 1000 described herein can contain aprocessing unit 1200. The processing unit 1200 can include one or moreendogenous, synthetic, or otherwise modified signal transductionpathway. In synthetic or otherwise modified signal transduction pathwaysat least one molecule involved in the signal transduction pathway can berecombinant, or otherwise non-natural. The signal transduction pathwaycan be a MAPK pathway, adenylyl cyclase pathway, phospholipase Cmediated pathway (e.g. inositol 1,4,5-triposphate (IP₃)/Diacyl glycerol(DAG) pathway), arachidonic acid pathway, cyclic AMP (cAMP) pathway,RhoGEF signaling pathways, ion channels (e.g. G-protein-regulatedrectifying K+ channels, P/Q- and N-type voltage gated channels,posphoinositide-3-kinase pathways, 6-arrestin, G protein-coupledreceptor kinases, histidine-specific protein kinase mediated pathways,tyrosine kinase mediate pathways, AKT pathways FAK mediated pathways,GSK3β pathways. In some embodiments, the processing unit 1200 cancontain molecules within the mating pathway of yeast. In other words,the GPCR of the sensing unit 1100 can be configured to stimulatemolecules in the mating pathway of yeast (e.g. Ste4, GPA1, Ste20, Ste5,Ste11, Ste7, and/or Fus3).

The signal transduction pathway of the processing unit 1200 can regulateone or more transcription factors. Regulation of transcription factorscan include, but is not limited to, activation or suppression oftranscription factors. One of ordinary skill in the art will appreciatethe myriad of ways activation or suppression of a transcriptionfactor(s) can occur and all are within the spirit and scope of thisdescription. The transcription factor can be native to the host cell. Inother embodiments, the transcription factor is a synthetic transcriptionfactor that is not native to the host cell or the signaling pathwayemployed by the processing unit 1200. In some embodiments, thetranscription factor is Ste12. In other embodiments, the transcriptionfactor is a synthetic transcription factor including, but not limitedto, STF1 (a transcription factor composed of the STE12 phosphorylationdomain and the Gal4 activation and DNA binding domains (Pi, H. W.,Chien, C. T., and Fields, S. (1997). Transcriptional activation uponpheromone stimulation can be mediated by a small domain of Saccharomycescerevisiae Ste12p, Mol Cell Biol 17, 6410-6418.)), STF2 (a transcriptionfactor composed of the STE12 phosphorylation domain, the synthetic B42activation domain and the bacterial LexA DNA binding domain (Golemis, E.A., and Brent, R. (1992) Fused Protein Domains Inhibit DNA-Binding byLexa, Mol Cell Biol 12, 3006-3014 and Peralta-Yahya, P., Carter, B. T.,Lin, H. N., Tao, H. Y., and Comish, V. W. (2008) High-ThroughputSelection for Cellulase Catalysts Using Chemical Complementation, J AmChem Soc 130, 17446-17452)), STF3 (a transcription factor composed ofthe CRE protein activation and phosphorylation domain with the Gal4 DNAbinding domain), STF4 (a transcription factor composed of the CREprotein activation and phosphorylation domain and the LexA DNA bindingdomain). The synthetic transcription factor can be configured tointeract with an endogenous or a synthetic promoter.

Response Unit

The GPCR-based chemical biosensor 1000 can contain a response unit 1300.The response unit can contain a signal molecule promoter operativelycoupled to a signal molecule gene, where the signal molecule geneencodes or otherwise (e.g. by activating other pathways in the cell thatresults in the production of a gene product, such as a protein that canbe measured) generates a signal molecule. The promoter can be configuredto stimulate or extinguish transcription of the signal molecule gene(and subsequent production of the signal molecule) upon binding orunbinding of a transcription factor (such as one stimulated by theprocessing unit 1200). In this way, as signal (either appearance ordisappearance of the signal molecule) can be generated by the GPCR-basedchemical biosensor 1000 in response to binding, unbinding, or otherinteraction of a chemical 1400 with the GPCR of the sensing unit 1100.

The transcription factor can be native to the host cell or synthetic. Inother embodiments, the transcription factor is a synthetic transcriptionfactor that is not native to the host cell or the signaling pathwayemployed by the processing unit 1200. In some embodiments, thetranscription factor is Ste12. In other embodiments, the transcriptionfactor is a synthetic transcription factor including, but not limitedto, STF1 (a transcription factor composed of the STE12 phosphorylationdomain and the Gal4 activation and DNA binding domains (Pi, H. W.,Chien, C. T., and Fields, S. (1997). Transcriptional activation uponpheromone stimulation can be mediated by a small domain of Saccharomycescerevisiae Ste12p, Mol Cell Biol 17, 6410-6418.)), STF2 (a transcriptionfactor composed of the STE12 phosphorylation domain, the synthetic B42activation domain and the bacterial LexA DNA binding domain (Golemis, E.A., and Brent, R. (1992) Fused Protein Domains Inhibit DNA-Binding byLexa, Mol Cell Biol 12, 3006-3014 and Peralta-Yahya, P., Carter, B. T.,Lin, H. N., Tao, H. Y., and Comish, V. W. (2008) High-ThroughputSelection for Cellulase Catalysts Using Chemical Complementation, J AmChem Soc 130, 17446-17452)), STF3 (a transcription factor composed ofthe CRE protein activation and phosphorylation domain with the Gal4 DNAbinding domain), STF4 (a transcription factor composed of the CREprotein activation and phosphorylation domain and the LexA DNA bindingdomain). The transcription factor can be directly stimulated by theprocessing unit or can be the product of another signal transductionpathway stimulated by the processing unit (via a transcription factor orother mode of pathway stimulation).

In some embodiments the transcription factor can have a sequence about90% to 100% identical to SEQ ID NOS: 51-53.

(STF1 sequence) SEQ ID NO: 51ATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGCGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCTAGACCATCTAGTACAACAAAATCAGATAATTCGCCTCCAAAATTAGAAAGCGAGAATTTTAAGGATAATGAGTTGGTAACAGTAACTAATCAGCCGCTTTTAGGCGTTGGCCTCATGGATGACGATGCGCCAGAATCCCCCTCTCAAATTAATGATTTTATTCCTCAGAAATTGATTATAGAACCCAATACTCTCGAATTGAATGGTCTCACAGAAGAAACGCCTCATGACTTACCCAAGAATACCGCTAAGGGCAGAGACGAAGAAGATTTTCCTCTCGACTATTTTCCTGTATCTGTTGAATACCCTACGGAGGAAAATGCGTTTGATCCGTTCCCTCCACAGGCTTTTACGCCAGCTGCCCCTTCCATGCCTATTTCCTATGATAACGTGAATGAAAGGGATTCTATGCCCGTTAATTCTCTTCTTAATAGATACCCCTATCAGTTATCAGTGGCACCCACTTTCCCAGTGCCACCATCATCATCGAGGCAACATTTTATGTATCCTTACGACGTTCCAGATTATGCTATTGACTCTGCAGCTCATCATGATAACTCCACAATTCCGTTGGATTTTATGCCCAGGGATGCTCTTCATGGATTTGATTGGTCTGAAGAGGATGACATGTCGGATGGCTTGCCCTTCCTGAAAACGGACCCCAACAATAATGGGTTCTAA (STF2 sequence) SEQ ID NO: 52ATGGGTGCTCCACCTAAGAAGAAAAGAAAGGTTGCCAAAGCTTTGACTGCCAGACAACAAGAAGTCTTCGATTTGATTAGAGATCATATTTCTCAAACTGGTATGCCACCAACTAGAGCTGAAATTGCTCAAAGATTGGGTTTCAGATCTCCAAACGCCGCTGAAGAACACTTGAAAGCTTTGGCTAGAAAGGGTGTCATTGAAATTGTTTCTGGTGCTTCTAGAGGTATTAGATTGTTGCAAGAAGAAGAAGAAGGTTTGCCATTGGTTGGTAGAGTCGGTAGACCATCTTCTACTACTAAATCTGATAACTCTCCACCAAAGTTGGAATCTGAAAACTTCAAAGATAACGAATTGGTTACTGTTACAAATCAACCATTGTTAGGTGTCGGTTTGATGGATGACGATGCTCCAGAATCTCCTTCTCAAATTAACGATTTCATTCCACAAAAGTTGATTATTGAACCAAACACTTTGGAATTGAACGGTTTGACTGAAGAAACTCCACACGATTTGCCAAAGAATACTGCCAAAGGTAGAGATGAGGAAGACTTCCCATTGGATTACTTTCCAGTTTCTGTCGAATATCCAACTGAAGAAAACGCTTTCGATCCATTTCCACCACAAGCCTTTACTCCAGCTGCACCATCTATGCCAATTTCTTACGATAACGTTAATGAAAGAGATTCTATGCCAGTCAACTCATTGTTGAATAGATACCCATATCAATTGTCTGTTGCTCCAACTTTCCCAGTTCCTCCATCTTCTTCAAGACAACACTTTATGGGTATTAACAAGGATATTGAGGAATGTAATGCCATCATTGAACAATTCATCGATTACTTGAGAACTGGTCAAGAAATGCCAATGGAAATGGCCGATCAAGCCATTAACGTTGTCCCAGGTATGACTCCAAAGACTATTTTGCACGCTGGTCCACCAATTCAACCAGATTGGTTGAAATCTAACGGTTTCCACGAAATTGAAGCTGATGTCAATGACACATCTTTGTTATTGTCTGGTGATGCCTCTTAA (STF3 sequence)SEQ ID NO: 53ATGACTATGGATTCTGGTGCTGATAATCAACAATCTTCTTGTAAAGATTTGAAAAGATTGTTTTCTGGTACTCAAATTTCTACTATTGCTGAATCTGAAGATTCTCAAGAATCTGTTGATTCTGTTACTGATTCTCAAAAAAGAAGAGAAATTTTGTCTAGAAGACCATCTTATAGAAAAATTTTGAATGATTTGTCTTCTATTGAACAAGCTTGTGATATTTGTAGATTGAAAAAATTGAAATGTTCTAAAGAAAAACCAAAATGTGCTAAATGTTTGAAAAATAATTGGGAATGTAGATATTCTCCAAAAACTAAAAGATCTCCATTGACTAGAGCTCATTTGACTGAAGTTGAATCTAGATTGGAAAGATTGGAACAATTGTTTTTGTTGATTTTTCCAAGAGAAGATTTGGATATGATTTTGAAAATGGATTCTTTGCAAGATATTAAAGCTTTGTTGACTGGTTTGTTTGTTCAAGATAATGTTAATAAAGATGCTGTTACTGATAGATTGGCTTCTGTTGAAACTGATATGCCATTGACTTTGAGACAACATAGAATTTCTGCTACTTCTTCTTCTGAAGAATCTTCTAATAAAGGTCAAAGACAATTGACTGTTTCTATTGATTCTGCTGCTCATCATGATAATTCTACTATTCCATTGGATTTTATGCCAAGAGATGCTTTGCATGGTTTTGATTGGTAA

The signal molecule promoter can be a native promoter in the host cell.Suitable native promoters include without limitation those involved inthe yeast mating pathway (e.g. FIG. 1 and Fus 1, Fig 3, Fig2, Fig4). Thesignal molecule promoter can be a synthetic promoter. Suitable syntheticpromoters include without limitation PGal4(5×), which is described ingreater detail elsewhere herein, and contains five Gal4 binding sitesand PLexA(4×), which is described in greater detail elsewhere herein,and contains 4 LexA binding sites. In some embodiments, the syntheticpromoter can have a nucleotide sequence about 90% to 100% identical toany one of SEQ ID Nos: 54-56

PGal4(5x): (The underlined ATG is the start codon) SEQ ID: NO: 54CCGAGCTCTTACGCGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGTCGAGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGTCGACTCTAGAGGGTATATAATGPLexA(4x): (The underlined ATG is the start codon) SEQ ID: NO: 55CCGAGCTCTTACGCGGGTCGAAGTGCTGTATATACTCACAGCAAGTGGAGTACTGTCCTCCGAGAACTGTATATACACCCAGGGAGTCGAGGGTCGAAGTACTGTATGAGCATACAGTAAGTGGAGTACTGTCCTCCGAGAACTGTATATAAATACAGTTAGTCGACTCTAGAGGGTATAT AATG(PCRE) SEQ ID NO: 56TCCTGGAAGTCTCATGGAGATTATACTTTATGCACCAGACAGTGACGTCAGCTGCCAGATCCCATGGCCGTCATACTGTGACGTCTTTCAGACACCCCATTGACGTCAATGGGAGAACTTTAGTATCCGTTTAGCTAGTTAGTACCTTTGCACGGAAATGTATTAATTAGGAGTATATTGAGAAATAGCCGCCGACAAAAAGGAAGTCTCATAAAAGTGTCTAACAGACAATTAGCGCAATAAGAAGAAAGAAAACGGATTGAAGTTGAGTCGAGAATAATATGGCACCCAGAAAACGCTTTAGGCTACTCGAATTAGGGTCACCAATGIn some embodiments, the promoter can be or include a repressor element.In some embodiments the repressor can consist of or include a sequenceabout 90% to about 100% identical to SEQ ID NO: 57 and/or SEQ ID NO: 58.

(pGal4(5x) repressor) SEQ ID NO: 57TCGACTCTAGAGGGTATATACCGAGCTCTTACGCGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGTCGAGGGTCGAAGCGGAGTACTGTCCTCCGAGTGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGGGATCCATG(pLexA(4x) repressor) SEQ ID NO: 58CCGAGCTCTTACGCGGGTCGAAGTGCTGTATATACTCACAGCAAGTGGAGTACTGTCCTCCGAGAACTGTATATACACCCAGGGAGTCGAGGGTCGAAGTACTGTATGAGCATACAGTAAGTGGAGTACTGTCCTCCGAGAACTGTATATAAATACAGTTAGTCGACTCTAGAGGGTATAT AATGATG

The signal molecule promoter can be operatively coupled to a signalmolecule gene, which can encode a suitable signal molecule. Suitablesignal molecules include without limitation, a fluorescent protein,β-galactosidase protein, a luciferase protein, and chloramphenicolacetyltransferase, antibiotic resistance markers such as KanMX4,auxotrophic genes such as His3, Ura3, TRp1, Leu2 which enable selectionsand counter selections, a biosynthetic gene or pathway that results inthe production of a colorimetric or fluorescent compound, such aslycopene, indigo or violacein, a synthetic RNA, a synthetic DNA or aribozyme Suitable fluorescent proteins include without limitations,green fluorescent proteins and enhanced green fluorescent proteins,yellow fluorescent proteins and enhanced yellow fluorescent proteins,blue fluorescent proteins and enhanced blue fluorescent proteins, cyanfluorescent proteins and enhanced cyan fluorescent proteins, orangefluorescent proteins and enhanced orange fluorescent proteins, and redfluorescent proteins and enhanced red fluorescent proteins. Fluorescentproteins are generally known in the art and are commercially available.All of these are within the scope and spirit of the present disclosure.The signal molecule gene can be codon optimized for expression withinthe particular host cell. In some embodiments, the signal molecule is afast maturing signal molecule.

Amplification Unit

As shown in FIGS. 2A and 2B, the GPCR-based chemical biosensor 1000 canoptionally contain an amplification unit 2000. The amplification unit2000 can be configured to directly (FIG. 2A) or indirectly (FIG. 2B)amplify the signal generated by the response unit 1300. Generally, theamplification unit 2000 can act as a feed forward loop that stimulatesincreased signal production from the response unit 1300 when theresponse unit is biologically acted upon, either directly or indirectly,by the processing unit 1200. In short, the amplification unit canautoamplify the signal from the response unit 1300 in response to achemical 1400 binding, unbinding, or otherwise interacting with the GPCRof the sensing unit 1100.

As shown in FIG. 2A the amplification unit 2000 can be configured todirectly amplify the signal generated by the response unit 1300. Theamplification unit can contain an amplification unit promoter that canbe operatively coupled to a transcription factor gene. The product(s) ofthe transcription factor gene can be a suitable transcription factor. Inoperation, the transcription factor stimulated by the processing unit1200 can bind the signal molecule gene promoter of the sensing unit 1300and the promoter of the amplification unit 2000. The promoter of theamplification unit 2000 can drive gene expression of a transcriptionfactor that can bind also bind or otherwise interact with the signalmolecule promoter of the response unit 1300 to drive gene expression ofthe signal molecule and generate additional signal molecules in a feedforward fashion. Insofar as additional signal molecules can be generatedwithout additional input stimulation from the sensing unit 1100, thesignal from the GPCR-based chemical biosensor can be amplified.

Suitable transcription factors produced by the amplification unit 2000can be any transcription factor configured to bind or otherwise activethe signal molecule gene promoter of the response unit 1300 and generatean upregulation in gene expression of the signal molecule gene. In someembodiments, the transcription factor produced by the amplification unit2000 can be the same transcription factor produced or stimulated by theprocessing unit 1200. In some embodiments, the transcription factorproduced by the amplification unit 2000 can be Ste12, STF1, or STF2.

Suitable promoters for the amplification unit 2000 can include nativeand synthetic promoters. The amplification unit promoter can be a nativepromoter in the host cell. Suitable native promoters include withoutlimitation those involved in the yeast mating pathway (e.g. FIG. 1 andFus 1, Fig3, Fig4, Fig2). The amplification unit promoter can be asynthetic promoter.

Suitable synthetic promoters include without limitation PGal4(5×), whichis described in greater detail elsewhere herein, and contains five Gal4binding sites and PLexA(4×), which is described in greater detailelsewhere herein, and contains 4 LexA binding sites. In someembodiments, the synthetic promoter can have a nucleotide sequenceidentical to any one of SEQ ID Nos: 54-56.

As shown in FIG. 2B, the optional amplification unit 2000 can indirectlyamplify the signal generated by the GPCR-based chemical biosensor. Inthese embodiments, the amplification unit 2000 can contain a firstamplification unit promoter operatively coupled to an intermediateactivator gene. The intermediate activator gene can encode for asuitable intermediate molecule that is capable of binding a secondamplification unit promoter that is operatively coupled to atranscription factor gene. Any promoter described herein or any othernative promoter can be used as a promoter in the amplification unit. Thepromoter can be operatively coupled to the transcription factor gene.The second amplification unit promoter and the transcription factor genecan be as described with respect to the amplification unit promoter andtranscription factor gene and gene product(s). Any promoter describedherein or any other native promoter can be used as a promoter in theamplification unit. The promoter can be operatively coupled to thetranscription factor gene or intermediate activator gene.

In operation, the transcription factor produced by the processing unit1200 can bind or otherwise activate both the signal molecule promoter ofthe response unit 1300 and the first amplification unit promoter. Whenthe first amplification unit promoter is activated it can driveexpression of the intermediate activator gene and thus production of asuitable intermediate activator molecule (e.g. another transcriptionfactor or other protein involved in up-regulation of genes, particularlythose that are part of the amplification unit). The intermediateactivator molecule can then bind or otherwise activate the secondamplification unit promoter and thus stimulate production of atranscription factor that can bind or otherwise interact with the signalmolecule promoter of the response unit 1300 to drive gene expression ofthe signal molecule and generate additional signal molecules in a feedforward fashion. Insofar as additional signal molecules can be generatedwithout additional input stimulation from the sensing unit 1100, thesignal from the GPCR-based chemical biosensor can be amplified.

Host Cells

The sensing unit, processing unit, and/or the response unit can beexpressed or otherwise contained within a single host cell. The hostcell can be eukaryotic or prokaryotic. In some embodiments, the hostcell can be a mammalian cell, a fungal cell, or a bacterial cell. Insome embodiments the host cell is a yeast cell. Suitable yeast speciesfor the host cell include but are not limited to S. cerevisiae, PichiaPastoris, Saccharomyces Pombe. Suitable strains of S. cerevisiaeinclude, but are not limited to the W303 strain (ATCC), PPY62, PPY58,PPY140, and PPY161. The GPCR-based chemical biosensors can be introducedinto the host cell via a single or multiple plasmid system or integratedinto the genome. The GPCR-based chemical biosensor or can be stably ortransiently expressed within the host cell. In some embodiments, thehost cell is different from a producer cell (i.e., a cell that producesa chemical to be detected by the GPCR-based chemical biosensor). TheGPCR-based chemical sensors can be used to evolutionary engineer orhigh-throughput engineering chemical-producing microbes usingmedium-throughput methods (e.g. 96-well plate), or high-throughputmethods (e.g. microfluidic chip).

Systems and Methods of Using the GPCR-Based Chemical Biosensors

Also described herein are systems and methods of using the GPCR-basedchemical biosensors. As described above the modular components of theGPCR-based chemical biosensors can be expressed within a host cell (alsoreferred to herein as a sensor cell or sensor strain). The host cell canthen be used in a method to sense a chemical (which includes proteins)of interest. The method can include incubating a host cell containing aGPCR-based chemical biosensor as described herein in a solution orenvironment containing a sample, a cell, or other composition to beanalyzed for a period of time. After the period of time, a suitableassay or other suitable measurement technique can be performed tomeasure the amount of signal molecule produced by the GPCR-basedchemical biosensor. One of skill in the art will appreciate that theparticular assays or measurement technique used will depend on the typeof signaling molecule produced. Suitable assays and measurementtechniques include, but are not limited to, flow cytometry, FACS,luciferase assays (single and dual), β-galactosidase assays, microtiterplate reader, and CAT assays, antibiotic selection, auxotrophic forwardand counter selection. Other assays and techniques will be readilyappreciated by those of ordinary skill in the art.

In some embodiments, the sensor cell or strain can be used to detect amedium chain fatty acid in a sample. In other embodiments the sensorstrain can be used to detect production of a desired chemical (whichincludes proteins) such as a medium chain fatty acid, from a producercell. These can be accomplished in a low-throughput or mediumthrough-put fashion. As shown in FIG. 3, producing strains can beanalyzed in a medium through put fashion by arraying different producingstrains in a multi-well plate, incubating the producing strains for afirst period of time to allow for generation of a chemical product fromthe producing strain(s).

After the first period of time, a sensor cell(s) can be added to thewells as desired and incubated for a second period of time to allow forinteraction, such as binding, between the chemical produced by theproducer cell in each well and the GPRC of sensor cell present in thesame well. The second period of time can be an amount of time sufficientfor biosensor production. The second period of time can range from about0 to about 96 hours, about 96 to about 72 hours, about 72 to about 60hours, about 60 hours to about 48 hours, about 48 hours to about 36hours, about 36 hours to about 24 hours, about 24 hours to about 12hours, about 12 hours to about 6 hours, about 4 hours to about 6 hours,about 2 hours to 4 hours, and about 0 to about 2 hours. In someembodiments, particularly those when a fast maturing signaling moleculeis used, the second period of time can range from about 0 hours to about3 hours. In other embodiments, the second period of time can be about 4hours. In further embodiments, the second period of time can be about 1hour.

After the second period of time, a suitable assay or measurementtechnique can be performed to measure the amount of signal moleculeproduced from each well. This can allow for determining which producingcells produced the chemical of interest. In embodiments, where thesignal measurement assay/technique can allow for quantification of theamount of signal produced, it can be determined which producing cellsproduced the most chemical. Such techniques that can allow forquantification include flow cytometry, FACS, luciferase assays,6-galactosidase assays, microtiter plate reader, antibiotic selection,auxotrophic forward and counter selection and CAT assays. Others will beappreciated by those of skill in the art. In this way, one can selectwhich producing strain is desired based on the determination of theirability to produce (or not produce) a particular chemical.

In some embodiments the sensor cells as described herein can be used inany of the methods previously described to detect a fatty acid. In someembodiments, the fatty acid is a medium chain fatty acid. In someembodiments, the medium chain fatty acid is a C10 fatty acid. In someembodiments, the GPCR-based chemical biosensor or assay using theGPCR-based chemical biosensor can have a linear range of detection of upto about 250 μM. The GPCR-based chemical biosensor can have a lineardetection range of about 500 μM or greater. In some embodiments thelinear detection range can be from about 0 to 1M or any range withinthat. In some embodiments, the GPCR-based chemical biosensor or assayusing the GPCR-based chemical biosensor can have a linear range ofdetection of up to about 500 μM. In some embodiments, the GPCR-basedchemical biosensor or assay using the GPCR-based chemical biosensor canhave a linear detection range of about 34 μM to about 250 μM. TheGPCR-based chemical biosensor or assay using the GPCR-based chemicalbiosensor can have a linear detection range of about 110 μM to about 500μM. The dynamic range of the GPCR-based chemical biosensor or assayusing the GPCR-based chemical biosensor can range from about 4 to about68. The dynamic range is the ratio of the highest fluorescence obtainedby the sensor in the presence vs. the absence of the chemical. It willbe appreciated that the linear and dynamic range can be customized basedon the configuration sensor unit, response unit, processing unit, andamplification unit, both individually and collectively as a system.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1 GPCR-Based Biosensors to Detect Medium Chain Fatty Acids

To rapidly construct chemical biosensors, GPCRs were exploited as thesensing unit. GPCRs can bind a large variety of chemicals from biogenicamines and carbohydrates to lipids and odors. GPCR-based chemicalsensors have been previously engineered in the yeast Saccharomycescerevisiae, as this organism is amenable to heterologous GPCRexpression. Although in the 1990s and 2000s GPCRs were commonly coupledto the yeast mating pathway to discover new ligands for known GPCRs,since then GPCR-based chemical sensing in yeast has been limited. Fourmain obstacles have hindered GPCR-based sensing in yeast: 1) theunsystematic expression of functional heterologous GPCRs on the yeastcell surface; 2) the unreliable coupling of heterologous GPCRs to theyeast mating pathway; 3) the poor functional expression of mammalianGPCRs; particularly olfactory GPCRs, such that only two olfactoryreceptors (rat ORI7 and human OR17-40) have been functionally expressedin yeast and have been used as the scaffold to express the ligandbinding domain of other olfactory receptors (ORL829, ORL451, MOR226-1);and 4) the weak signal strength of the biosensor.

Here, GPCR codon optimization and the use of a wide array of yeastpromoters and plasmids were used overcome these obstacles and enable therapid construction of GPCR-based chemical sensors in yeast.Specifically, using a plug-and-play strategy, sensing (GPCR), processing(signaling pathway), and response units (transcriptionfactor/promoter/reporter gene) can be mixed and matched to predictablygenerate chemical sensors (see e.g. FIGS. 1, 2, 7, 12, and 18).

In this Example, the rapid construction of GPCR-based yeast sensors todetect saturated medium-chain fatty acids is demonstrated. Fatty acidsare the immediate precursors to the advanced biofuels fatty acid methylesters (FAMEs), which can serve as a “drop in” replacement for D2diesel. FAMEs derived from medium-chain fatty acids (C8-C12) have bettercold properties than traditional canola oil-derived (C16-C22) FAMEs.Microbial production of medium-chain fatty acids is a challengingproblem both in S. cerevisiae and Escherichia coli, with titers reachingless than 100 mg/L, a stark contrast to the titers reached for C16-C18in E. coli (5 g/L) and S. cerevisiae (400 mg/L).

A medium-chain fatty acid sensor could be used for the engineering ofmicrobes with improved medium-chain fatty acid production or thedetection of medium-chain fatty acids in a sample. In this Example, thesignal after activation of the endogenous Ste2/α-factor was measured todetermine the upper limit for future GPCR-based chemical sensors thatwould rely on heterologous GPCR sensing units coupling to the yeastmating pathway. Then, two GPCRs known to bind fatty acids in mammaliancells were each coupled to the yeast mating pathway to form two separatesystems. One of the GPCR-based sensors reliably detects C8-C12 fattyacids with a 13- to 17-fold increase in signal after activation. Thesensor is specific to medium-chain fatty acids, not being able to detectlong-chain fatty acids or medium-chain aldehydes, alcohols or C10esters.

To engineer a GPCR-based chemical sensor strain, two genes in the yeastmating pathway were deleted to avoid cell cycle arrest (far1) and reducethe spontaneous rate of GPCR inactivation upon chemical sensing (sst2).Next, the dynamic range of the endogenous GPCR-based sensor(Ste2/α-factor) was determined using the mating pathway-dependenttranscription factor Ste12, which upregulates mating pathway genes (FIG.4). The response of the Ste2/α-factor sensor using GFP under control oftwo mating pathway promoters (P_(FUS1) and P_(FIG1)) from either asingle-copy (s) or a multi-copy (m) reporter plasmids (FIG. 5) wasobserved. The maximum x-fold increase in signal after activation,defined as the maximal GFP fluorescence in the presence of chemical overthe signal in the absence of chemical, for P_(FUS1) and P_(FIG1) from amulti-copy plasmid was observed to be 24- and 68-fold, respectively(P-value 0.009) (FIG. 6). The maximum x-fold increase in signal afteractivation for P_(FUS1) and P_(FIG1) from a single-copy plasmid was 20-and 40-fold, respectively, (P-value 0.014). The maximum x-fold increasein signal activation from P_(Fig1)-GFP (s) and P_(Fig1)-GFP (m) wasstatistically significant (P-value 0.009).

To generate a sensor to detect medium-chain fatty acids, Ste2 in themodified yeast strain having deletion of Far1 and Sst2 was replaced witha GPCR known to bind medium-chain fatty acids in mammalian cells andcoupled it to the yeast mating pathway with P_(FIG1)-GFP as the reporterplasmid, which resulted in GFP fluorescence upon medium-chain fatty acidaddition (FIG. 7). The GPCRs tested were the olfactory receptor OR1G1(Sanz, G., Schlegel, C., Pernollet, J. C., and Briand, L. (2005)Comparison of odorant specificity of two human olfactory receptors fromdifferent phylogenetic classes and evidence for antagonism, ChemicalSenses 30, 69-80) and the free fatty acid receptor GPR40 (Itoh, Y., etal. (2003) Free fatty acids regulate insulin secretion from pancreaticbeta cells through GPR40, Nature 422, 173-176). First, OR1G1 and GPR40sensing of even, medium-chain saturated fatty acids (C8-C16) usingP_(FIG1)-GFP(m) was tested. A reliable signal after fatty acid additionwas not observed (FIGS. 23 and 24). Hypothesizing that the largestandard deviation of P_(FIG1)-GFP(m) contributed to the inability todetect medium-chain fatty acids, P_(FIG1)-GFP(s) was tested as thereporter plasmid. Using P_(FIG1)-GFP(s) enabled the OR1G1 based-sensorto detect C8, C10 and C12 fatty acids with 13-, 17-, and 13-foldincreases in signal after activation, respectively (FIGS. 8-9). UsingP_(FIG1)-GFP(s) also enabled the GPR40 based-sensor to detect C8, C10and C12 saturated fatty acids, with 14-, 25-, and 16-fold increases insignal after activation, respectively (FIGS. 10-11). It was alsoexplored whether the OR1G1-based sensor signal could be improved byexpressing the OR1G1 from a single-copy (s) rather than a multi-copy (m)plasmid. Results are demonstrated in FIG. 25. OR1G1(m) and OR1G1(s) wereobserved to have similar increases in signal after activation with 500μM decanoic acid.

The OR1 G1-based sensor signal modification was carried out by using i)the mammalian olfactory G_(a) subunit (G_(olf)) that normally couples toOR1G1 instead of the yeast G_(a) (GPA1), and ii) a hybrid G_(α) subunitcomposed of GPA1 carrying the five C-terminal amino acids fromG_(olf)—both strategies having been previously successful to link GPCRsensing to the yeast mating pathway. Results are demonstrated in FIG.26. G_(α) subunit engineering did not significantly alter theOR1G1-based sensor signal. Using the chromosomal GPA1 resulted in thegreatest increase in signal after activation above 250 μM decanoic acid.

To confirm that the medium-chain fatty acids were signalling via theGPCR sensing unit and not through a different cellular mechanism, thechemical sensor strain in the presence and absence of the GPCRs andeither 0 or 500 μM decanoic acid was tested (FIG. 12). An increase inGFP fluorescence was only observed in the presence of both 500 μMdecanoic acid and either OR1G1 (P-value <0.001) or GPR40 (P-value<0.001) (FIG. 13).

To demonstrate that the chemical signal was transmitted via the yeastmating pathway, the mating pathway transcription factor Ste12 wasdeleted. The sensor strain having the three deletions (Far 1, Sst2, andSte12) was then tested in the presence and absence of the GPCRs andeither 0 or 500 μM decanoic acid. There was no observable increase inGFP fluorescence in the absence of Ste12 and the presence of both GPCRand 500 μM decanoic acid (FIG. 14). Interestingly, deletion of Ste12 wasobserved to produce greater overall GFP background fluorescence, whichcan be attributed to transcription factors other than Ste12, such as theTATA box binding protein, binding to the pheromone response elements inP_(Fig1)-GFP(s). Taken together, these data demonstrate that decanoicacid is sensed by the heterologous GPCR, which uses the yeast matingpathway as the processing unit and not a different cellular mechanism.

To determine the specificity of the OR1G1- and the GPR40-based sensors,we tested the ability of the sensors to detect saturated C8, C10 and C12fatty aldehydes, important targets for the perfume industry, saturatedC8, C10 and C12 fatty alcohols, important targets for the detergentindustry, as well as C10 fatty acid methyl- and ethyl-esters, which areadvanced biofuels that can serve as replacements for D2 diesel wastested. Results for OR1G1-based sensors are demonstrated in FIGS.15A-15C. Results for GPR40-sensors are demonstrated in FIGS. 16A-16C.Except for C10 aldehyde, the OR1G1 based-sensor was unable to detectaldehydes, alcohols or esters with more than a 3-fold increase in signalafter activation. The GPR40 based-sensor detected the C10 aldehyde at125 μM and C12 aldehyde at 250 μM, both with a 3-fold increase in signalafter activation, but was unable to detect the C8 aldehyde. Similarly tothe OR1G1 based-sensor, the GPR40 based-sensor was unable to detectalcohols or esters with more than a 3-fold increase in signal afteractivation. These data demonstrate that the OR1G1- and GPR40-basedsensors are specific to medium chain fatty acids.

Example 2 GPCR-Based Biosensors Containing a Synthetic Response Unit

The biosensors of Example 1 were modified by introducing a syntheticresponse unit capable of taking information from the yeast matingpathway and exclusively activating green fluorescent protein (GFP)expression, resulting in a decanoic acid sensor with a 30-fold increasein signal after activation. Introduction of the synthetic response unitalso altered the linear range of the sensor. To improve the biosensorresponse to medium-chain fatty acids, the endogenous mating pathwaytranscription factor Ste12, which activates more than 100 mating pathwaygenes, was bypassed.

To engineer a system in which medium-chain fatty acid sensing wouldtrigger only GFP transcription, Ste12 was replaced in the yeast straincarrying deletions of Ste2, Sst2, and Far1 with one of two synthetictranscription factors (STFs): 1) STF1, which is composed of the Ste12phosphorylation domain and the Gal4 activation and DNA binding domains(Pi, H. W., Chien, C. T., and Fields, S. (1997) Transcriptionalactivation upon pheromone stimulation mediated by a small domain ofSaccharomyces cerevisiae Ste12p, Mol Cell Biol 17, 6410-6418), and 2)STF2, which is composed of the Ste12 phosphorylation domain, thesynthetic B42 activation domain and the bacterial LexA DNA bindingdomain (Golemis, E. A., and Brent, R. (1992) Fused Protein DomainsInhibit DNA-Binding by Lexa, Mol Cell Biol 12, 3006-3014 andPeralta-Yahya, P., Carter, B. T., Lin, H. N., Tao, H. Y., and Comish, V.W. (2008) High-Throughput Selection for Cellulase Catalysts UsingChemical Complementation, J Am Chem Soc 130, 17446-17452). STF1 canactivate transcription of GFP placed under control of a syntheticminimal promoter carrying five Gal4 DNA binding sites (P_(Gal4(5×))).STF2 activates transcription of GFP placed under control of a syntheticminimal promoter carrying four lexA DNA binding sites (P_(LexA(4×)))(FIGS. 17 and 18).

Under glucose conditions, STF1 triggers only P_(Gal4(5x))-GFP expressionas endogenous galactose promoters are repressed by Mig1. STF2 triggersonly expression of P_(LexA(4x))-GFP as lexA binding sites are ofprokaryotic origin and orthogonal to the yeast machinery. Coupling ofthe STF1/P_(Gal4(5×))-GFP response unit to the OR1G1 based-sensorresulted in a 30-fold increase in signal after activation in thepresence of 800 μM decanoic acid (FIGS. 19 and 20), which is almost a200% improvement over the Ste12/P_(FIG1)-GFP(s) response unit (Example1). Further, the OR1G1 based-sensor coupled to the STF1/P_(Gal4(5×))-GFPresponse unit also was observed to have an improved linear range,reaching to 500 μM decanoic acid when compared to the OR1G1 based-sensorcoupled to the Ste12/P_(FIG1)-GFP(s) response unit, in which linearrange plateaued at 250 μM decanoic acid. Coupling of theSTF2/P_(LexA(4×))-GFP response unit to the OR1G1 based-sensor resultedin only a 7-fold increase in signal after activation in the presence of800 μM decanoic acid. Coupling of the STF1/P_(Gal4(5×))-GFP responseunit to the GPR40 based-sensor unit resulted in a 28-fold increase insignal after activation in the presence of 500 μM decanoic acid (FIGS.21-22), though this increase was not statistically significant whencompared to coupling the Ste12/P_(FIG1)-GFP(s) response unit to theGPR40-based sensor (25-fold increase). Coupling the GPR40 based-sensorto STF2/P_(LexA(4x)) showed only a 4-fold increase in GFP expressionupon decanoic acid exposure.

Example 3 Determining the Dynamic Range of GPCRs of Examples 1 and 2

The linear and dynamic range and linear range, binding affinity andsensitivity of the sensors was evaluated to determine their utility inchemical screening applications. This is believed to be the first reportof a whole-cell biosensor for medium-chain fatty acids and the firstcoupling of a synthetic response unit to a GPCR-based yeast sensor forthe sensing of non-endogenous chemicals. The rapid generation ofnon-invasive chemical sensors such as the ones presented in this workwill be important to the future engineering of chemical-producingmicrobes.

Dose response curves of the GPCR-based sensors in the presence of fattyacids were fitted to the Hill equation (Table 1). Response curves forthe OR1G1- and GPR40-based sensors could be fitted to transfer functionsfor all saturated fatty acids. For the detection of decanoic acid withthe OR1G1-based sensor, changing the response unit fromSte12/P_(FIG1)-GFP(s) to STF1/P_(Gal4(5×))-GFP was observed to improvethe dynamic range from a 17- to a 30-fold increase, change the linearrange from 34-250 μM to 110-500 μM, and increase the K_(M) from 65 μM to248 μM. Further, the sensitivity of the response to decanoic acid wasalso observed to increase from n=2.3 to n=3.2. For the detection ofdecanoic acid with the GPR40-based sensor, changing the response unitfrom Ste12/P_(FIG1)-GFP(s) to STF1/P_(Gal4(5x))-GFP did not result in astatistically significant change in dynamic range, but was observed tochange the linear range from 36-100 μM to 47-250 μM and increase theK_(M) from 69 μM to 114 μM. Therefore, by simply changing the responseunit, the dynamic and linear range of GPCR-based sensors can be alteredwithout the need for using a GPCR with a different binding affinity forthe compound of interest. This can be a significant advantage over themodular GPCR system described herein over currently available singlecomponent sensor. Sensors with different dynamic and linear ranges maybe useful to different applications. For example, when the engineeringof a chemical producing microbe is optimized, it can be desirable tohave a sensor for different production levels, i.e. one sensor with alinear range from 10-100 uM, another one from 100 to 500 uM, etc.

TABLE 1 GFP Hill Reporter max Dynamic Linear K_(M) coeff. Strain plasmidGPCR TF Chemical (AU) range range (μM) (n) W303 P_(Fig1)- Ste2 Ste12 αfactor 2314 40 8-50 nM 0.03 1.7 Δfar1, GFP(s) Δsst2 P_(Fus1)- Ste2 Ste12α factor 4565 20 2-25 nM 0.02 1.1 GFP(s) P_(Fus1)- Ste2 Ste12 α factor20525 24 5-25 nM 0.02 1.8 GFP(m) P_(Fig1)- Ste2 Ste12 α factor 7566 684-25 nM 0.02 1.4 GFP(m) W303 P_(Fig1)- OR1G1 Ste12  C8 acid 239 1319-250 μM 230 1.5 Δfar1, GFP(s) OR1G1 Ste12 C10 acid 308 17 34-250 μM 652.25 Δsst2 OR1G1 Ste12 C12 acid 233 13 1-250 μM 50 0.85 Δste2 P_(Fig1)-GPR40 Ste12  C8 acid 197 14 36-250 μM 162 2.25 GFP(s) GPR40 Ste12 C10acid 339 25 36-100 μM 69 4.1 GPR40 Ste12 C12 acid 222 16 2-250 μM 1480.7 W303 P_(Gal4(5x))- OR1G1 STF1 C10 acid 1126 30 110-500 μM 248 3.2Δfar1, GFP(m) GPR40 STF1 C10 acid 573 28 47-250 μM 114 3 Δsst2,P_(LexA(4x))- OR1G1 STF2 C10 acid 405 7 2-100 μM 69 0.62 Δste2, GFP(m)GPR40 STF2 C10 acid 501 4 4-100 μM 62 0.77 Δste12

In Table 1: ^(a)Dose response curves were fitted to the Hill equation toderive the biosensor transfer functions from which the performancefeatures were obtained. TF: transcription factor. GFPmax is the highestfluorescence obtained by the sensor in the presence vs the absence ofthe chemical. Dynamic range is the ratio of the highest fluorescenceobtained by the sensor in the presence vs the absence of the chemical.Linear range is the series of chemical concentrations for which a changein signal can be detected by the sensor. The minimum limit of the linearrange is estimated as the chemical concentration corresponding to 10%signal saturation from the fitted model. KM is the chemicalconcentration at half maximal signal, estimated by linear interpolationfrom experimental data. Hill coefficient (n) is the sensitivity of thesystem.

The ability of the OR1G1-GPRC-based biosensors to detect medium-chainfatty acids, such as decanoic acid, contain within a mixture of otherfatty acids was evaluated. The results are shown in FIG. 27. Theinability for the OR1G1-GPRC-based biosensor to detect decanoic acid ina mixture with C14 and C16 acids may be due to the toxicity of fattyacids to cell growth (see FIGS. 28A-28C). Transforming decanoic acidconcentration to titers, the OR1G1-sensor coupled to theSte12/P_(FIG1)-GFP(s) response unit can detect decanoic acid titers fromabout 6 to about 43 mg/L. The OR1G1-based sensor coupled to theSTF1/P_(Gal4(5×))-GFP response unit to detect decanoic acid titers fromabout 19 to about 86 mg/L. Since extracellular decanoic acid productionin E. coli is about 80 mg/L (Choi, Y. J., and Lee, S. Y. (2013)Microbial production of short-chain alkanes, Nature 502, 571-574) and S.cerevisiae's is about 3 mg/L (Leber, C., and Da Silva, N. A. (2014)Engineering of Saccharomyces cerevisiae for the Synthesis of Short ChainFatty Acids, Biotechnol Bioeng 111, 347-358) the OR1G1 sensor has theappropriate linear range to screen for decanoic acid-producing formicrobes with increased titers. In another application, the sensors canbe used as a systems biology tools to interrogate less engineeredstrains for alternative routes to increase fatty acid production in amedium-throughput fashion (10³ samples/day). Such a throughput wouldallow the screening of entire transposon libraries, or simply existingmicrobial deletion collections, such as those from S. cerevisiae or E.coli for the discovery of novel regulatory elements that affect thedecanoic acid production.

Example 4 Experimental Methods for Examples 1-3

Yeast Strain Construction.

The yeast haploid strain W303 (MATa, leu2-3, 112 trp1-1 can1-100 ura3-1ade2-1 his3-11,15) was used in this study. The open reading frames (ORF)of Far1 Sst2, Ste2, and Ste12 were deleted using Delitto perfetto(Storici, F., Lewis, L. K. & Resnick, M. A. In vivo site-directedmutagenesis using oligonucleotides. Nature biotechnology 19, 773-776,(2001) and Stuckey, S. & Storici, F. Gene Knockouts, in vivoSite-Directed Mutagenesis and Other Modifications Using the DelittoPerfetto System in Saccharomyces cerevisiae. Method Enzymol 533,103-131, (2013)). For all deletions the core cassette CORE-I-Scelcontaining the I-Scel gene under control of the inducible P_(GAL1)promoter, as well as the hygromycin resistant maker and a counterselectable K. lactis URA3 marker gene were used. For W303 Δfar1 the corecassette was amplified from pGSHU with primers KM1/KM2 and used todelete the Far1 ORF. The cassette was subsequently popped out usingprimers KM7/KM8 to create strain PPY62. For W303 Δfar1, Δsst2, the corecassette was amplified from pGSHU with primers KM9/KM10 and used todelete the Sst2 ORF in PPY62. The cassette was subsequently popped outusing primers KM13/KM14 to create strain PPY58. For W303 Δfar1, Δsst2,Δste2, the core cassette was amplified from pGSHU with primers KM59/KM60and used to delete the Ste2 ORF in PPY58. The cassette was subsequentlypopped out using primers KM61/KM62 to create strain PPY140. For W303Δfar1, Δsst2, Δste2, Δste12, KanMX4 was amplified from pFA6a-KanMX4 withprimers KM49/KM50 and used to delete the Ste12 ORF in PPY140 to createstrain PPY161. Some yeast strains and plasmids used are listed in Table2. Note-I combined Table 1 from the ACS Synthetic biology 2014 paperwith the Table provided in the Supplement of the ACS Synthetic biology2014 paper in Table 2 below.

TABLE 2 Plasmids and Yeast Strains Plasmid/base Reference strain wherewhere Strain # applicable Description applicable PPY39 pESC-Leu2pESC-Leu2, P_(Gal1), P_(Gal10) Agilent PPY34 pESC-His3 pESC-His3,P_(Gal1), P_(Gal10) Agilent PPY15 pRS415-Leu2 YE-type (episomal) shuttlevector ATCC PPY13 pRS413-His3 YE-type (episomal) shuttle vector ATCCPPY38 pGFP Enhanced GFP Storici's Lab PPY43 pKM43 pESC-Leu2-P_(Gal1)-GFPThis study PPY96 pKM96 pESC-Leu2-P_(Fus1)-GFP This study PPY97 pKM97pESC-Leu2-P_(Fig1)-GFP This study PPY111 pKM111pESC-His3-P_(tef1)-p_(adh1) This study PPY144 pKM144pESC-His3-P_(TEF1)-P_(ADH1)-STF1 This study PPY150 pSTF1 Commerciallysynthesized Gal4_(AD)- Pi et al.⁴ Ste12(P)-Gal4_(DBD) PPY185 pOR1G1Commercially synthesized GPCR OR1G1 This study codon optimized for S.cerevisiae PPY194 pSTF2 Commercially synthesized B42_(AD)- This studySte12(P)-LexA_(DBD) PPY269 pKM269 pESC-His3-P_(TEF1)-OR1G1- P_(ADH1)This study PPY282 pGPR40 commercially synthesized GPCR GPR40 This studycodon optimized for S. cerevisiae PPY389 pKM389 pRS415-Leu2-P_(Fus1)-GFPThis study PPY469 pKM469 pESC-His3-P_(TEF1)-GPR40- P_(ADH1) This studyPPY470 pG_(olf) commercially synthesized G_(olf) sequence PPY513 pKM513pESC-His3-P_(TEF1)-OR1G1- P_(ADH1)-G_(olf) This study PPY528 pKM528pESC-Leu2-P_(Gal4(5x))-GFP This study PPY566 pFA6a-KanMX4 G418 resistantgene (KanMx4) Storici's Lab PPY571 pGSHU CORE-I-SceI cassette includingthe I-SceI Storici's Lab gene under the inducible GAL1 promoter, thehygromycin resistant gene and counter selectable KIURA3 marker genePPY586 pKM586 pRS415-Leu2-P_(Fig1)-GFP (s) This study PPY595 pKM595pESC-His3-P_(TEF1)-OR1G1-P_(ADH1)-STF1 This study PPY637 pGAL4(5x)Commercially synthesized minimal This study promoter with five GAL4 DNAbinding sites PPY651 pKM651 pESC-His3-P_(TEF1)-OR1G1-P_(ADH1)-Gpa1-G_(olf) This study PPY684 pKM684pRS413-His3-P_(Tef1)-OR1G1 This study PPY685 pKM685pESC-His3-P_(TEF1)-GPR40-P_(ADH1)-STF1 This study PPY686 pKM686pRS413-His3-P_(TEF1)-OR1G1- P_(ADH1)- Gpa1- This study G_(olf) PPY690pLexA Commercially synthesized LexA DNA This study binding sites PPY712pKM712 pESC-Leu2-P_(LexA(4x))-GFP This study PPY727 pKM727 pESC-His3-P_(TEF1)-OR1G1-P_(ADH1)-STF2 This study PPY728 pKM728pESC-His3-P_(TEF1)-GPR40-P_(ADH1)-STF2 This study PPY11 W303 MATa,leu2-3, trp1-1, can1-100, ura3-1, ATCC ade2-1, his3-11 PPY62 PPY11 Δfar1This study PPY58 PPY11 Δfar1, Δsst2 This study PPY140 PPY11 Δfar1,Δsst2, Δste2 This study PPY161 PPY 11 Δfar1, Δsst2, Δste2, Δste12 Thisstudy PPY638 PPY 58 pESC-Leu2-PFus1-GFP This study PPY639 PPY58pESC-Leu2-PFig1-GFP This study PPY640 PPY 58 pRS415-Leu2-PFus1-GFP Thisstudy PPY641 PPY 58 pRS415-Leu2-PFig1-GFP This study PPY653 PPY 58pESC-Leu2 This study PPY654 PPY 58 pRS415-Leu2 This study PPY643 PPY140pESC-His3-PTEF1-OR1G1, pRS415- This study Leu2-PFig1-GFP PPY644 PPY140pESC-His3-PTEF1-GPR40, pRS415-Leu2- This study PFig1-GFP PPY912 PPY140pRS13-His3-PTEF1-OR1G1, pRS415- This study Leu2-PFig1-GFP PPY913 PPY140pESC-His3-PTEF1-OR1G1-PADH1-Golf, This study pRS415-Leu2- PFig1-GFPPPY914 PPY140 pESC-His3-PTEF1-OR1G1-PADH1- This study GPA1-Golf, pRS415-Leu2-PFig1-GFP PPY915 PPY140 pRS13-His3-PTEF1-OR1G1-PADH1- This studyGPA1-Golf, pRS415- Leu2-PFig1-GFP PPY656 PPY140 pESC-His3, pRS415-Leu2This study PPY916 PPY140 pRS13-His3, pRS415-Leu2 This study PPY794PPY140 pESC-His3, pRS415-Leu2-PFig1-GFP This study PPY795 PPY161pESC-His3-PTEF1-OR1G1, pRS415- This study Leu2-PFig1-GFP PPY832 PPY161pESC-His3-PTEF1-GPR40, pRS415-Leu2- This study PFig1-GFP PPY657 PPY161pESC-His3, pESC-Leu2 This study PPY833 PPY161 pESC-His3,pRS415-Leu2-PFig1-GFP This study PPY661 PPY161pESC-His3-PTEF1-OR1G1-PADH1-STF1, This study pESC-Leu2- PGal4(5x)-GFPPPY818 PPY161 pESC-His3-PTEF1-OR1G1-PADH1-STF2, This study pESC-Leu2-PLexA(4x)-GFP PPY796 PPY161 pESC-His3-PTEF1-GPR40-PADH1-STF1, This studypESC-Leu2, PGal4(5x)-GFP PPY819 PPY161 pESC-His3-PTEF1-GPR40-PADH1-STF2,This study pESC-Leu2- PLexA(4x)-GFP

Vector Construction.

Enhanced GFP was amplified from pEGFP using primers KM19/KM20 and clonedunder P_(Gal1) in pESC-Leu2 at BamHI/HindIII to createpESC-Leu2-P_(Gal1)-GFP (pKM43). To construct pESC-Leu2-P_(Fus1)-GFP(pKM96) and pESC-Leu2-P_(Fig1)-GFP (pKM97), the Fus1 and FIG. 1promoters were amplified from the W303 genome using primers KM15/KM56and KM54/KM55, respectively. The Fus1 and Fig1 promoters were clonedinto pKM43 at NotI/BamHI. To construct pRS415-Leu2-P_(Fus1)-GFP (pKM389)and pRS415-Leu2-P_(Fig1)-GFP (pKM586), P_(Fus1)-GFP and P_(Fig1)-GFPwere amplified from the pKM96 or pKM97 using primers KM159/KM160 orKM185/KM186 and cloned into pRS415-Leu2 at HindIII/BamHI. To constructpESC-His3-P_(Tef1)-P_(Adh1) (pKM111), the Tef1 and Adh1 promoters wereamplified from the W303 yeast genome using primers KM27/KM28 andKM23/24, respectively, and cloned into pESC-His3 at BamHI/NotI usingSLIC (Li, M. Z. & Elledge, S. J. Harnessing homologous recombination invitro to generate recombinant DNA via SLIC. Nature methods 4, 251-256,(2007)). To construct pESC-His3-P_(Tef1)-GPR40-P_(Adh1) (pKM469), GPR40was amplified from pGPR40 using primers SB89/SB90 and cloned into pKM111at BamHI/SacII. To construct pESC-His3-P_(Tef1)-OR1G1-P_(Adh1) (pKM269),OR1G1 was amplified from pOR1G1 using primers SB3/SB4 and cloned intopKM111 at BamHI/SacII. To construct pRS413-His3-P_(Tef1)-OR1G1 (pKM684)P_(Tef1)-OR1G1 was amplified from pKM269 using primers KM251/KM252 andcloned in pRS413-His3 between NotI/BamHI. To constructpESC-His3-P_(Tef1)-OR1G1-P_(Adh1)-Golf (pKM513) G_(olf) was amplifiedfrom pG_(olf) using primers KM245/KM246 and cloned in pKM269 atNotI/SpeI. To construct pESC-His3-P_(Tef1)-OR1G1-P_(Adh1)-Gpa1-Golf(pKM651) GPA1 was amplified from W303 genomic DNA using primersKM191/KM192 and cloned in pKM269 at NotI/SpeI. To constructpRS413-His3-P_(Tef1)-OR1G1-P_(Adh1)-Gpa1-Golf (pKM686)P_(Tef1)-OR1G1-P_(Adh1)-Gpa1-G_(olf) was amplified from pKM651 usingprimers KM193/KM194 and cloned in pRS413 at NotI/BamHI. To constructpESC-His3-P_(Tef1)-P_(Adh1)-STF1 (pKM144), STF1 was amplified from pSTF1using primers KM43/KM44 and cloned into pKM111 at NotI/SpeI. Toconstruct pESC-His3-P_(Tef1)-OR1G1-P_(Adh1)-STF1 (pKM595) STF1 wasamplified from pKM144 using primers KM189/KM190 and cloned in pKM269 atNotI/SpeI. To construct pESC-Leu2-P_(Gal4(5x))-GFP (PPY528), five Gal4binding sites were amplified from pGal4(5×) using primers KM187/KM188and cloned into pESC-Leu2 at BamHI/NotI. To constructpESC-His3-P_(Tef1)-GPR40-P_(Adh1)-STF1 (pKM685) STF1 was amplified frompKM144 using primers KM189/190 and cloned in pKM469 at NotI/SpeI. Toconstruct pESC-His3-P_(Tef1)-OR1G1-P_(Adh1)-STF2 (pKM727) STF2 wasamplified from pSTF2 using primers KM197/KM198 and cloned in pKM269 atNotI/SpeI. To construct pESC-His3-P_(Tef1)-GPR40-P_(Adh1)-STF2 (pKM728)STF2 was amplified from pSTF2 using primers KM197/KM198 and cloned inpKM269 at NotI/SpeI. To construct pESC-Leu2-P_(LexA(4×))-GFP (pKM712)pLexA was amplified from pLexA using primers KM195/KM196 and cloned inpKM43 at NotI/BamHI. A list of primers and their sequences are shown inTable 3.

TABLE 3 SEQ ID NO: Name Sequence 1 KM1GTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTATAGGGATAACAGGGTAAT TTCGTACGCTGCAGGTCGAC 2 KM2AAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCC GAACTACCGCGCGTTGGCCGATTCAT3 KM7 CCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTATAGTTCGGGAATCGAGGCCCGTATTTCGAGGCTTTTGCTT 4 KM8AAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTATAATAGATTGCCTTCTTACGCCCACGAAGCTTTCCAGTGG 5 KM9TATCTGAGGCGTTATAGGTTCAATTTGGTAATTAAAGATAGAGTTGTAAGTAGGGATAACAGGGTAAT TTCGTACGCTGCAGGTCGAC 6 KM10AGGACTGTTTGTGCAATTGTACCTGAAGATGAGTAAGACTCTCAA TGAAACCGCGCGTTGGCCGATTCAT7 KM13 GTTATAGGTTCAATTTGGTAATTAAAGATAGAGTTGTAAGTTTCATTGAGAGTCTTACTCATCTTCAGGTACAATTGCAC 8 KM14GTGCAATTGTACCTGAAGATGAGTAAGACTCTCAATGAAACTTACAACTCTATCTTTAATTACCAAATTGAACCTATAAC 9 KM19CGTCAAGGAGAAAAAACCCCGGATCCATGGTGAGCAAGGGCGA GGA 10 KM20TCTTAGCTAGCCGCGGTACCAAGCTTTTACTTGTACAGCTCGTC CA 11 KM15TGTAATCCATCGATACTAGTGCGGCCGCACGATGATTCAGTTCG CCTT 12 KM23TGTAATCCATCGATACTAGTGCGGCCGCTGTATATGAGATAGTT GATT 13 KM24TTTTGAAGCTATGGTGTGTGATCCTTTTGTTGTTTCCGGG 14 KM27CCTATAGTGAGTCGTATTACGGATCCTTTGTAATTAAAACTTAGA T 15 KM28AGCTAGCCGCGGTACCAAGC 16 KM43AATCAACTATCTCATATACAGCGGCCGCATGAAGCTACTGTCTTC TAT 17 KM44CATCCTTGTAATCCATCGATACTAGTTTAGAACCCATTATTGTTG G 18 KM49CTTTTATAGCGGAACCGCTTTCTTTATTTGAATTGTCTTGTTCACC AAGGATGGGTAAGGAAAAGACTCA19 KM50 CTGGCCCGCATTTTTAATTCTTGTATCATAAATTCAAAAATTATATTATATTAGAAAAACTCATCGAGCA 20 KM54TGTAATCCATCGATACTAGTGCGGCCGCATCACCCTGCATTGCC TCTT 21 KM55TCCTCGCCCTTGCTCACCATGGATCCTTTTTTTTTTTTTTTTTTGT 22 KM56TCCTCGCCCTTGCTCACCATGGATCCTTTGATTTTCAGAAACTTG A 23 KM59AATTGGTTACTTAAAAATGCACCGTTAAGAACCATATCCAAGAATCAAAATAGGGATAACAGGGTAATTTCGTACGCTGCAGGTCGAC 24 KM60ACCTTATACCGAAGGTCACGAAATTACTTTTTCAAAGCCGTAAAT TTTGACCGCGCGTTGGCCGATTCAT25 KM61 TTAAAAATGCACCGTTAAGAACCATATCCAAGAATCAAAATCAAAATTTACGGCTTTGAAAAAGTAATTTCGTGACCTTC 26 KM62GAAGGTCACGAAATTACTTTTTCAAAGCCGTAAATTTTGATTTTGATTCTTGGATATGGTTCTTAACGGTGCATTTTTAA 27 KM159TCGAGGTCGACGGTATCGATAAGCTTACGATGATTCAGTTCGCC TT 28 KM160GCGGCCGCTCTAGAACTAGTGGATCCCTTCGAGCGTCCCAAAA CCT 29 KM185CCCCCCTCGAGGTCGACGGTATCGATAAGCTTATCACCCTGCAT TG 30 KM186CGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCTTCGAGCGT CCC 31 KM187TAATCCATCGATACTAGTGCGGCCGCCCGAGCTCTTACGCGGGT CG 32 KM188TCCTCGCCCTTGCTCACCATGGATCCTATATACCCTCTAGAGTC GA 33 KM189CATCCTTGTAATCCATCGATACTAGTTTAGAACCCATTATTGTTG G 34 KM190TCAACTATCTCATATACAGCGGCCGCATGAAGCTACTGTCTTCTA T 35 KM191CATCCTTGTAATCCATCGATACTAGTTCACAACAATTCGTACTGT A 36 KM192TCAACTATCTCATATACAGCGGCCGCATGGGGTGTACAGTGAGT AC 37 KM193TCGAATTCCTGCAGCCCGGGGGATCCGAGCGACCTCATGCTAT ACC 38 KM194TGGAGCTCCACCGCGGTGGCGGCCGCCTTCGAGCGTCCCAAAA CCT 39 KM195TAATCCATCGATACTAGTGCGGCCGCCCGAGCTCTTACGCGGGT CG 40 KM196TCCTCGCCCTTGCTCACCATGGATCCCATTATATACCCTCTAGAG T 41 KM197CATCCTTGTAATCCATCGATACTAGTTTAAGAGGCATCACCAGAC A 42 KM198TCAACTATCTCATATACAGCGGCCGCATGGGTGCTCCACCTAAG AA 43 KM245CATCCTTGTAATCCATCGATACTAGTTTACAACAATTCGTACTGTT 44 KM246TCAACTATCTCATATACAGCGGCCGCATGGGTTGCCTGGGTAAT TC 45 KM251TCGAATTCCTGCAGCCCGGGGGATCCCACACACCATAGCTTCAA AA 46 KM252TGGAGCTCCACCGCGGTGGCGGCCGCCTTCGAGCGTCCCAAAA CCT 47 SB3ATCTAAGTTTTAATTACAAAGGATCCATGCATCACCATCACCATC 48 SB4TTAGAGCGGATCTTAGCTAGCCGCGGTTATGGGGAATGAATCTT TC 49 SB89TTAGAGCGGATCTTAGCTAGCCGCGGTTACTTTTGAGATTTACCA CC 50 SB90TAAGTTTTAATTACAAAGGATCCAAAACAATGGATTTGCCACCAC AATT

Autofluorescense Strains.

For the Ste2p/α-factor sensor, W303 Marl, Asst2 carrying a blank plasmid(pRS15-Leu2) with the same marker and copy number as the reporterplasmid (pRS15-Leu2-P_(FIG1)-GFP) was used to measure cellautofluorescence. For the OR1G1- and GPR40-sensors, W303 far1Δ sst2Δste2Δ carrying pRS15-Leu2 and a blank plasmid (pESC-His3) with the samemarker and copy number as the GPCR plasmid (pESC-His3-P_(TEF1)-GPCR) wasused. For the synthetic response unit experiments, W303 far1Δ sst2Δste12Δ carrying pRS15-Leu2 and pESC-His3 was used.

Biosensing Protocol.

For the Ste2/α-factor sensor, strains PPY638, PPY639, PPY640, PPY641were grown overnight in synthetic complete media with 2% glucose andlacking leucine (SD glu (L⁻)). The next day, the cells were used toinoculate a 20 mL of SD glu (L⁻) to an OD₆₀₀=0.06 and incubated for 18hrs at 30° C. (150 r.p.m.). The cells were centrifuged, re-suspended in5 mL SD glu (L⁻), used to innoculate 5 ml of fresh SD glu (L⁻) toOD₆₀₀=0.6. α-factor (0-100 nM, Zymo Y1001) was added to the medium andincubated for 4 hrs at 30° C. (150 r.p.m.) before reading for cellfluorescence using a flow cytometer. For the OR1G1- and GPR40-basedsensors using Ste12/P_(FIG1)-GFP response unit, strains PPY643, PPY644were grown overnight in SD glu and lacking histidine and leucine (SD glu(HL⁻)). The next day, the cells were used to inoculate a 20 mL of SD glu(HL⁻) to an OD₆₀₀=0.06 and incubated for 18 hrs at 15° C. (150 r.p.m.).The cells were centrifuged, re-suspended in 2 ml SD glu (HL⁻) toOD₆₀₀=0.6. C8, C10, C12, C14 and C16 saturated fatty acids (0-800 μM)were added to the medium and incubated for 4 hrs at 30° C. (150 r.p.m.)before reading for cell fluorescence using a flow cytometer. For theOR1G1-based sensor expressed from a single copy plasmid, and whencoupled to G_(olf) and GPA1-Golf, strains PPY912, PPY913, PPY914, andPPY915 were processed using the same protocol as the OR1G1-based sensorusing Ste12/P_(FIG1)-GFP response unit. For the OR1G1- and GPR40based-sensors with synthetic response units, PPY661, PPY796, PPY818,PPY819, were processed using the same protocol as the OR1G1- and GPR40based-sensors using the Ste12/P_(FIG1)-GFP response unit. All fattyacids were dissolved in DMSO and the final concentration of DMSO in thecultures was 1%. GFP fluorescence was measured using BD LSRII flowcytometers with the following settings: 488 nM laser line, 515-545 nmfilter, FSC: 178 volts, SSC: 122 volts, FITC: 600 volts. Fluorescencedata was collected from 10,000 viable cells for each experiment. Flowcytometry histogram analysis was done using FlowJo software.

Statistical Analysis.

For all experiments, cell autofluorescence, measured using the biosensorstrain with empty plasmids, was subtracted from the fluorescence of thebiosensor at all chemical concentrations to obtain GFP fluorescenceattributable to the sensor. Maximum x-fold increase in signal afteractivation is defined as the quotient of GFP fluorescence in thepresence and absence (0 μM) of the chemical. Standard deviation for theX-fold increase in GFP fluorescence was calculated using:

Δz=z SQRT [(Δx/x)²+(Δy/y)²]

where x and Δx are the average fluorescence and standard deviation inthe absence of the chemical, respectively, and y and Δy are the averagefluorescence and standard deviation in the presence of the chemical,respectively. Z and Δz are x-fold increase in signal activation and itsstandard deviation, respectively.

Biosensor Performance Calculations.

The Hill equation was used to fit the transfer function to derive thebiosensor performance features:

GFP=GFP₀+(GFP_(chemical)−GFP₀)(x^(n)/K_(M) ^(n)+x^(n))

where GFP₀ is the fluorescence in the absence of chemical,GFP_(chemical) is the fluorescence in the presence of the chemical, x isthe ligand concentration, K_(M) is the ligand concentration that resultsin half-maximal signal, and n is a measure of the biosensor sensitivity(Hill coefficient). The K_(M) value was determined directly from theexperimental data while the n value is the best fit to the experimentaldata using Matlab Curve-Fitting Toolbox and the Hill equation. Weestimated from our fitted model, the substrate concentrationcorresponding to 10% of signal saturation as the lower bound of thelinear range of the sensor.

Example 6 GPCR-Based Biosensors Using a Fast Maturing FluorescentReporter Protein

To increase the speed and signal strength of the sensor, we have testeda number of different fluorescent proteins that have a faster maturationtime and higher intrinsic fluorescence. Five different fluorescentproteins were tested and their activity was measured and in the sensorcontext and compared it to enhanced GFP (EGFP), which was usedpreviously. Specifically, the superfolder GFP, GFPγ, mCherry, mKate2,and Venus were tested.

Biosensing Protocol

The Ste2/α-factor sensor strain (PPY58) transformed with either plasmidspRP973, pRP974, pRP975, pRP976, pRP977, pRP984, pRP985, pRP986 weregrown overnight in synthetic complete media with 2% glucose and lackingleucine (SD glu (L⁻)). The next day, the cells were used to inoculate a20 mL of SD glu (L⁻) to an OD₆₀₀=0.06 and incubated for 18 hrs at 30° C.(150 r.p.m.). The cells were centrifuged, re-suspended in 5 mL SD glu(L), used to innoculate 5 ml of fresh SD glu (L⁻) to OD₆₀₀=0.6. α-factor(100 nM, Zymo Y1001) was added to the medium and incubated for 4 hrs at30° C. (150 r.p.m.) before reading for cell fluorescence using a flowcytometer

Vector Construction.

Super folder GFP was amplified from pPPY875(pFA6-Link-yoSuperfolderGFP-caURA3) using primers RP1/RP2 and clonedunder P_(Fig1) in KM97 (pESC-Leu2-P_(Fig1)-GFP, multi copy plasmid) atBamHI/HindIII to create pESC-Leu2-P_(Fig1)-Superfolder GFP (pRP973). Toconstruct pESC-Leu2-P_(Fig1)-Gamma GFP (pRP974) gammaGFP was amplifiedfrom pPPY874 (pFA6-Link-yoGammarGFP-spHis5) using primers RP3/RP4 andcloned under and P_(Fig1) in pKM97 at BamHI/HindIII to ceratepESC-Leu2-P_(Fig1)-gammaGFP (pRP974). mCherry was amplified from pKM945using primers RP5/RP6 and cloned under P_(Fig1) in KM97 at BamHI/HindIIIto create pESC-Leu2-P_(Fig1)-mCherry (pRP975). mKate2 was amplified frompPPY889(pDONR P4-P1R-mKate2) using primers RP7/RP8 and cloned underP_(Fig1) in KM97 at BamHI/HindIII to create pESC-Leu2-P_(Fig1)-mKate2(pRP976). Venus was amplified from pPPY873(pKT0090) using primersRP9/RP10 and cloned under P_(Fig1) in KM97 at BamHI/HindIII to createpESC-Leu2-P_(Fig1)-Venus (pRP977).

To clone into pRS415 (single copy plasmid) P_(Fig1)-Superfolder GFP wasamplified from pRP973 using primers KM296/297 and cloned at BamHI andNotI to create pRS415-Leu2 P_(Fig1)-superfolder GFP (pRP984).P_(Fig1)-Gamma GFP was amplified from pRP974 using primers KM296/297 andcloned at BamHI and NotI to create pRS415-Leu2-P_(Fig1)-gamma GFP(pRP985). P_(Fig1)-mKate2 was amplified from pRP976 using primersKM296/297 and cloned at BamHI and NotI to createpRS415-Leu2-P_(Fig1)-mKate2 (pRP 986).

TABLE 4 Primers used in example 4 SEQ Primer ID NO Sequence RP1 76ACAAACAAAAAAAAAAAAAAAAAAGGATCCATGACAGTCAACACTAAGAC RP2 77CGGATCTTAGCTAGCCGCGGTACCAAGCTTTTATAATTGGCCAGTCTTTTT C RP3 78ACAAACAAAAAAAAAAAAAAAAAAGGATCCATGGGTAGGAGGGCTTTTG RP4 79CGGATCTTAGCTAGCCGCGGTACCAAGCTTTTACAACACTCCCTTCGTG RP5 80ACAAACAAAAAAAAAAAAAAAAAAGGATCCATGGTGAGCAAGGGCGAG RP6 81CGGATCTTAGCTAGCCGCGGTACCAAGCTTTTATAATTTGGACTTGTACAG C RP7 82ACAAACAAAAAAAAAAAAAAAAAAGGATCCATGGTGAGCGAGCTGATTA RP8 83CGGATCTTAGCTAGCCGCGGTACCAAGCTTTTATCTGTGCCCCAGTTTG RP9 84ACAAACAAAAAAAAAAAAAAAAAAGGATCCATGTCTAAAGGTGAAGAATTAT RP10 85CGGATCTTAGCTAGCCGCGGTACCAAGCTTTTATTTGTACAATTCATCCAT AC KM296 86TCGAATTCCTGCAGCCCGGGGGATCCATCACCCTGCATTGCCTCTT KM297 87TGGAGCTCCACCGCGGTGGCGGCCGCCTTCGAGCGTCCCAAAACCT KM304 88ACAAAAAAAAAAAAAAAAAAGGATCCATGAAAGTCCAAATAACCAA KM305 89TCTTAGCTAGCCGCGGTACCAAGCTTTCAGGTTGCATCTGGAAGGT

TABLE 5 Plasmids used in Example 4 Reference Strains Plasmid Fluorescentwhere number Name Copy Protein applicable PPY973 pRP973pESC-Leu2-P_(Fig1)- Superfolder This study sGFP GFP PPY974 pRP974pESC-Leu2- P_(Fig1)- GFPγ This study GFPγ PPY975 pRP975 pESC-Leu2-P_(Fig1)- mCherry This study mCherry PPY976 pRP976 pESC-Leu2-P_(Fig1)-mKate2 This study mKate2 PPY977 pRP977 pESC-Leu2-P_(Fig1)- Venus Thisstudy Venus PPY984 pRP984 pRS-Leu2- P_(Fig1)- Superfolder This studysGFP GFP PPY985 pRP985 pRS-Leu2- P_(Fig1)- GFPγ This study GFPγ PPY986pRP986 pRS-Leu2- P_(Fig1)- mKate2 This study mKate2

Results are shown in Tables 6 and 7 and FIGS. 31 and 32.

TABLE 6 Brightness of different fluorescent proteins relative to EGFP.The brightness of each fluorescent protein was measured in S.cerevisiae. Brightness (% relative Class Protein to EGFP) ReferenceFar-red mKate2 74 Shcherbo D., et al. Biochem. J. 2009, 418, 567-574.Red mCherry 47 Shaner N C, et al. Nat. Biotechnol. 2004, 22, 1567-72.Yellow- Venus green Green EGFP 100 Green Superfolder 50 Lee S, et al.PloS one GFP 2013, 8. Green GFPγ 155 Lee S, et al. PloS one 2013, 8.

TABLE 7 Maturation half-time for different fluorescent proteins. Themethod to measuring maturation time is different for each fluorescentprotein. Maturation Organism Protein t_(0.5) (min) (measured) ReferencemKate2 <20 E. coli Shcherbo D., et al. Biochem. J. 2009, 418, 567-574.mCherry 16.9-30.3 S. cerevisiae Khmelinskii A, et al. Nat. Biotechnol.2012, 30, 708-14. Venus 11.2 ± 1.6 S. cerevisiae Ball, David A., et al.PloS ONE 2014, 9, e107087. EGFP  60 E. coli Sniegowski, J. A. et al.Biochem. Biophys. Res. Commun. 2005, 332, 657-663. Super- 5.63 ± 0.82 S.cerevisiae Khmelinskii A, et al. Nat. folder Biotechnol. 2012, 30,708-14. GFP GFPγ

Example 7 GPCR-Based Biosensors Having Amplified Signals

To increase the signal by the sensor upon chemical (e.g. decanoic acid)addition, the transcription factor (e.g. Ste12) that gets activated bythe signaling cascade (e.g. yeast mating pathway) and results in thetranscription activation of a fluorescent protein (e.g. GFP) andfluorescence, also drives the expression of the transcription activation(e.g. Ste12) itself, resulting in a feed forward loop and signalamplification. The plasmid for this feed forward set up(PESC-P_(Fig1)-Ste12) was constructed. Ste12 was amplified from W303genomic DNA using primers KM304/KM305 and cloned under P_(Fig1) in pKM97at BamHI and HindIII to create pESC-Leu2-P_(Fig1)-Ste12 (pKM1000).

Example 8 Repression of Response Unit Signaling in Response to ChemicalStimulus

Briefly, the yeast biosensor was configured to contain a PlexA(4×)repressor and STF2 utilizing the MAPK signaling cascade. The signalingmolecule was a GFP and signal in response to exposing the biosensor tovarying concentrations of decanoic acid was evaluated.

Example 9 Screening of Olfactory GPCRs for Use in GPCR-Based Biosensors

The OR1G1 GPCR that has been used to demonstrate sensing of decanoicacid was used to generate seven saturation mutagenesis libraries aroundthe active site with the goal of engineering this sensor to binddifferent biofuel molecules. The GPCR libraries are being screened withdifferent GPCR-based biosensors configured to detect biofuel and/orcomponents thereof. Below is a sequence obtained when one of thelibraries was tested against ethyl decanoate.

Below are the sequence modifications in the 7 saturation mutagenesislibraries using the GPCR OR1 G1 as the starting GPCR scaffold:

Library 1: Met 81, Pro 183, Asp 259, and Val 276 Library 2: Phe 104, Leu110, Pro 183, Val 276 Library 3: Lys 80, Phe 104, Leu 184, Phe 256Library 4: Met 105, Val 108, Phe 256, Val 276 Library 5: Met 105, Ser255, Phe 256, Val 276 Library 6: Lys 80, Met 81, Asn 84, Gln 100 Library7: Asp 191, Ser 255, Phe 256, Asp 259

OR1G1 wild type sequence SEQ ID NO: 59ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCATTCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA >A2 hit (nucleotide sequence) SEQ ID NO: 60ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGTCTTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACGTGTTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCATTCTGTGTCGGCTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCACTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA >A2 hit (amino acid sequence) SEQ ID NO: 61MHHHHHHEGKNLTSISECFLLGFSEQLEEQKPLFGSFLFMYLVTVAGNLLIILVIITDTQLHTPMYFFLANLSLADACFVSTTVPKSLANIQIQSQAISYSGCLLQLYFFMLFVMLEAFLLAVMAYDCYVAICHPLHYILIMSPGLCIFLVSASWIMNALHSLLHTLLMNSLSFCANHEIPHFFCDINVLLSLSCTDPFTNELVIFITGGLTGLICVLCLIISYTNVFSTILKIPSAQGKRKAFSTCSSHLSVVSLFFGTSFCVGFSSPSTHSAQKDTVASTMYTVVTPMLNPFIYSLRNQEIKSSLRKLIWVRKIHSP J6 (no mutations) SEQ ID NO: 62ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCATTCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA J8 (no mutations) SEQ ID NO: 63ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCATTCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L3 Cl0EE 4 SEQ ID NO: 64ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTTGCTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCCTCACTTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCTATATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L3 C12EE 5 (97 by insertion) SEQ ID NO: 65ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATGTACTTCAACATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATAGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCAGACTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTAAAGGATACCGTTGCTTCCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCACGCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAAL3 C12EE 6 (97bp insertion) SEQ ID NO: 66ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATGTACTTCAACATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATAGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCAGACTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTAAAGGATACCGTTGCTTCCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCACGCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L5 C10ME 2SEQ ID NO: 67ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTCCCTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCCACCTTTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCTAGATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L5 C10ME 3 SEQ ID NO: 68ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTGTCTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCCAGGATTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCTCGATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L5 C10ME 4 SEQ ID NO: 69ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTGTCTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCGCGGCTTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCAATATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L5 C10ME 6 SEQ ID NO: 70ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTATCTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCGCGGCTTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCAGGATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L5 C10ME 7 SEQ ID NO: 71ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTACCTTGCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTTCACGTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGTTCCAATATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L6 isobutanol 10 SEQ ID NO: 72ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATTTGGCTGTCATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGAATTTGTACTTCTTTATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCATTCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L6 isobutanol 12 SEQ ID NO: 73ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGAAGTTGGCTATGATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGTAGTTGTACTTCTTTATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCATTCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L7 C10EE 6 (no mutations) SEQ ID NO: 74ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTATGTTGTTCGTTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCGATCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCTCATTCTGTGTCGATTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA L7 C10EE 9 SEQ ID NO: 75ATGCATCACCATCACCATCACGAAGGTAAGAATTTGACCTCTATTTCCGAGTGTTTCTTACTTGGTTTCTCCGAACAATTGGAAGAACAGAAGCCATTGTTCGGTTCCTTCCTGTTTATGTACTTGGTCACCGTCGCTGGTAACTTGTTAATCATATTAGTCATTATTACCGATACCCAGTTACATACCCCAATGTATTTCTTCTTGGCTAACTTATCCCTAGCTGACGCTTGTTTCGTTTCCACTACCGTCCCAAAGATGTTGGCTAACATTCAAATTCAATCCCAAGCTATTTCCTACTCCGGTTGTTTATTGCAATTGTACTTCTTTATGTTGTTCGTATGTTGGAGGCTTTCTTGTTGGCTGTTATGGCTTACGATTGCTACGTCGCTATTTGTCATCCATTGCATTACATCTTGATTATGTCCCCAGGTTTGTGTATCTTCTTAGTCTCCGCCTCCTGGATTATGAACGCTTTGCATTCCTTGTTGCATACCTTGTTAATGAACTCTTTATCCTTCTGTGCTAACCACGAAATTCCACATTTCTTTTGTGATATTAACCCATTGTTGTCCTTGTCCTGTACCTCTCCATTCACCAACGAATTGGTTATCTTCATTACCGGTGGTTTGACTGGTTTGATTTGTGTCTTGTGTTTGATTATCTCCTACACTAACGTCTTCTCCACCATTTTGAAAATTCCATCCGCTCAAGGTAAAAGAAAAGCATTCTCCACCTGTTCCTCCCATTTGTCCGTTGTCTCCTTGTTCTTTGGTACCCCGAAGTGTGTCGCCTTCTCTTCCCCATCCACTCATTCCGCTCAAAAGGATACCGTTGCTTCCGTTATGTACACCGTCGTCACTCCAATGTTGAATCCATTTATTTATTCCTTAAGAAATCAAGAAATTAAGTCCTCCTTGAGAAAGTTGATTTGGGTTAGAAAGATTCATTCCCCATAA

We claim:
 1. A biosensor comprising: a sensing unit, the sensing unitcomprising: a G-protein coupled receptor (GPCR); a processing unit, theprocessing unit comprising: a signal transduction pathway; a responseunit, the response unit comprising: a recombinant signal molecule gene,where the recombinant signal molecule gene is operatively coupled to apromoter that is responsive to the processing unit, wherein the sensingunit is in biologic communication with the processing unit and theprocessing unit is in biologic communication with the response unit. 2.The biosensor of claim 1, wherein the GPCR is a heterologous GPCR. 3.The biosensor of claim 2, wherein the GPCR is codon optimized forexpression in yeast.
 4. The biosensor of claim 1, wherein the GPCR bindsa medium chain fatty acid.
 5. The biosensor of claim 1, wherein the GPCRis selected from the group consisting of: M3 muscarinic receptor, D2SDopamine receptor, Beta2 Adrenergic receptor, Beta Alanine Receoptor,Nicotinoamide receptor, OR56, Geosmin GPCR, melatonin receptor (mel1a),AT1R, OR1G1; GPR40, STE2, STE3, and a GPCR having an amino acid sequenceabout 90% to 100% identical to SEQ ID NO: 59-75.
 6. The biosensor ofclaim 1, further comprising an amplification unit, wherein theamplification unit is biologically coupled to the response unit.
 7. Thebiosensor of claim 1, wherein the signal transduction pathway includesthe transcription factor Ste12 or a synthetic transcription factor,where the synthetic transcription factor has a sequence about 90%-100%identical to SEQ ID NO: 51-53.
 8. The biosensor of claim 7, wherein thepromoter is Fig1, Fus1, or a synthetic promoter, wherein the syntheticpromoter has a sequence 90% to 100% identical to SEQ ID NO: 54-56. 9.The biosensor of claim 1, wherein the recombinant signal molecule is afluorescent protein.
 10. The biosensor of claim 9, wherein thefluorescent protein is a fast maturing fluorescent protein.
 11. Anengineered yeast cell comprising: a sensing unit, the sensing unitcomprising: a G-protein coupled receptor (GPCR); a processing unit, theprocessing unit comprising: a signal transduction pathway; a responseunit, the response unit comprising: a recombinant signal molecule gene,where the recombinant signal molecule gene is operatively coupled to apromoter that is responsive to the processing unit, wherein the sensingunit is in biologic communication with the processing unit and theprocessing unit is in biologic communication with the response unit. 12.The engineered yeast cell of claim 11, wherein the GPCR is capable ofbinding a medium chain fatty acid.
 13. The engineered yeast cell ofclaim 11, wherein the GPCR is selected from the group consisting of: M3muscarinic receptor, D2S Dopamine receptor, Beta2 Adrenergic receptor,Beta Alanine Receoptor, Nicotinoamide receptor, OR56, Geosmin GPCR,melatonin receptor (mel1a), AT1R, OR1G1; GPR40, STE2, STE3, and a GPCRhaving an amino acid sequence about 90% to 100% identical to SEQ ID NO:59-75.
 14. The engineered yeast cell of claim 11, further comprising anamplification unit, wherein the amplification unit is biologicallycoupled to the response unit.
 15. The engineered yeast cell of claim 11,wherein the signal transduction pathway includes the transcriptionfactor Ste12 or a synthetic transcription factor, wherein the synthetictranscription factor has a sequence about 90%-100% identical to SEQ IDNO: 51-53.
 16. The engineered yeast cell of claim 11, wherein thepromoter is Fig1, Fus1, or a synthetic promoter, wherein the syntheticpromoter has a sequence 90% to 100% identical to SEQ ID NO: 54-56. 17.The engineered yeast cell of claim 11, wherein the recombinant signalmolecule is a fluorescent protein.
 18. The engineered yeast cell ofclaim 11, wherein the yeast cell has a combination of gene deletionsselected from the group consisting of: (1) a Far1 deletion; (2) a Far1and a sst2 deletion; (3) a Far1 deletion, a sst2 deletion, and a Ste2deletion; and (4) a Far1 deletion, a sst2 deletion, a Ste2 deletion, anda ste12 deletion.
 19. A method comprising: contacting an engineeredyeast cell of claim 11 with a sample; and detecting a signal generatedby the response unit.
 20. The method of claim 19, wherein the step ofcontacting the engineered yeast cell as in claim 11 with a samplefurther comprises incubating the engineered yeast cell of claim 11 witha biofuel producing cell.